Abstract:
One aspect of the invention provides a method for fabrication of a membrane on a surface. The method includes: providing a surface interfacing two environments, wherein one of the environments is a liquid; providing a flow-recirculating fluidic device having channel exits in the liquid environment in proximity of the surface; and delivering locally one or more processing solutions. The one or more processing sources including one or more membrane sources adapted and configured to form a membrane on the surface.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation under 35 U.S.C. §120 of International Application No. PCT/IB2014/001089, filed Jan. 19, 2014, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/754,554, filed Jan. 19, 2013. The entire contents of each of these applications are hereby incorporated by reference herein. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    The most important class of two-dimensional fluids are biological membranes. Biomembranes consist of lipids and are the key components of all living organisms, defining walls of cells and organelles inside them, which allows separating different chemical cellular processes from each other. In order to fulfill transport and control requirements of life, these membranes host numerous membrane proteins, which act as gateways responsible for uptake of nutrients and transmitting chemical signals from and to the surrounding, to name a few examples. Besides membrane proteins, other micro- and nano-scale objects, ranging from vesicles, bacteria and viruses to synthetic nanoparticles, are of interest under the aspect of binding or associating to biological membranes. 
       SUMMARY OF THE INVENTION 
       [0003]    This invention provides a method and a system to locally create, modify and use two-dimensional fluid membrane structures on a surface. 
         [0004]    One aspect of the invention provides a method for fabrication of a membrane on a surface. The method includes: providing a surface interfacing two environments, wherein one of the environments is a liquid; providing a flow-recirculating fluidic device having channel exits in the liquid environment in proximity of the surface; and locally delivering one or more processing solutions. The one or more processing sources include one or more membrane sources adapted and configured to form a membrane on the surface. 
         [0005]    This aspect of this invention can have a variety of embodiments. In one embodiment, the flow-recirculating fluidic device is a microfluidic device. The channel exits can be positioned at a distance of about 10 μm to about 100 μm from the surface. 
         [0006]    The method can further include controlling a positioning device to facilitate translation of the channel exits relative to the surface. The positioning device can be adapted and configured to translate the channel exits to create a two-dimensional fluid membrane having a geometry defined by a translation path. The positioning device can be a micromanipulator. The positioning device can be a scanning stage. The positioning device can include an electronic controller adapted and configured to control speed and trajectory of translation. 
         [0007]    The processing solution can be switched between two or more solutions. Two or more different processing solutions can be delivered simultaneously. One of the processing solutions can be selected from the group consisting of: a chemical conjugation agent addressing a functional group in the membrane, a membrane soluble dye, and a fixation agent. One of the processing solutions can be a detergent adapted and configured to remove a portion of the membrane or its components from the surface. 
         [0008]    A gel can be formed near the surface and the gel adapted and configured to remove the membrane from the surface. 
         [0009]    Multiple membrane sources can be used to create the membrane with variable composition. Translation and switching between membrane sources can be synchronized, thereby creating spatially heterogeneous membrane geometries. Pulse width flow modulation can be used to create membranes with continuously variable composition. 
         [0010]    The membrane can be a surfactant multilayer, a surfactant single layer, a surfactant double layer, a double lipid bilayer, a single lipid bilayer, a lipid monolayer. The membrane can contain additional components. 
         [0011]    One of the processing solutions can be adapted and configured to modify the membrane locally. 
         [0012]    The membrane may be formed only when the membrane source is in direct contact with the surface. The membrane can be formed through fusion of the membrane source into an existing membrane. The membrane can be spatially heterogeneous in composition, and components of the membrane can be transported in the membrane by two-dimensional diffusion. 
         [0013]    The processing solution can be used to remove the membrane or parts of the membrane locally from the surface. Removed membrane components can be collected for analysis. 
         [0014]    The surface can be a solid-liquid interface. The surface can include one or more selected from the group consisting of: glass, metal, plastic, rubber, silicon, and oxides. 
         [0015]    The surface can be a gel-liquid interface. The surface can contain a pattern. The pattern can have different chemical or physical properties than non-patterned regions of the surface. The membrane can be deposited from the membrane source selectively on the pattern. The membrane may propagate exclusively on the pattern. 
         [0016]    Transport properties in the membrane can vary depending on location on the pattern. 
         [0017]    The membrane source can comprise surfactant micelles. 
         [0018]    The membrane source can comprise surfactant vesicles. The membrane source can comprises membrane extracted from biological cells. The vesicles can be small unilamellar vesicles. The vesicles can be multilamellar vesicles. 
         [0019]    The method can further comprise applying a field or gradient along some part of the membrane. The field can be a hydrodynamic flow field sufficient to impart a shear stress on the membrane. The field can be an electric field. The field can be a magnetic field. The gradient can be a thermal gradient. The gradient can be a chemical gradient. The field or gradient can cause the membrane to propagate along the surface. The field or gradient can cause one or more of the membrane components to migrate in the membrane. Migration can cause separation of two or more of the membrane components. The field or gradient can change direction along the membrane. The direction of the field can change at a stable stagnation point for membrane attached components. The stable stagnation point can be used to accumulate or trap membrane-attached components. 
         [0020]    The method can further include heating the membrane. Heat can be applied to a local region of the membrane. Heating can be applied globally. Heating can cause a change in membrane fluidity. Heating can cause a change in membrane adhesion strength. 
         [0021]    The method can further comprise analyzing the membrane. The analyzing step can utilize a sensor, electrochemical sensing, microscopy, spectroscopy, and/or total internal reflection. 
         [0022]    The surface can be a liquid-liquid interface. The surface can be a liquid-gas interface. The liquid can include water. The channel cross-sectional dimensions can be between about 10 μm and about 100 μm. 
         [0023]    The modification can be chemical or biological and can be selected from the group consisting of: a conjugation reaction, a cleavage reaction, dissociation, formation or breakage of covalent bond or coordination bond, nucleic acid hybridization, antigen-antibody recognition, and ion pairing. One or more additional components can be selected from the group consisting of: proteins, nanoparticles, microspheres, virus particles, vesicles, cell, bacterial cells, surfactant molecules, lipid molecules and non-lipid molecules. 
         [0024]    Another aspect of the invention provides a method of fabricating a membrane. The method includes: introducing a fluidic device into a volume of confining liquid such that one or more outlet ports of the fluidic device are positioned below an outer surface of the confining liquid; and dispensing one or more surfactant-containing liquids from the fluidic device into the confining liquid such that the one or more surfactant-containing liquids are hydrodynamically confined between the confining liquid and a substrate below the confining liquid, thereby fabricating a membrane. 
         [0025]    This aspect of the invention can have a variety of embodiments. The fluidic device can be a flow-recirculating fluidic device. The flow-recirculating fluidic device can include at least one inlet port. 
         [0026]    The confining liquid can be water or an aqueous solution. 
         [0027]    The one or more surfactant-containing liquids can include a suspension of vesicles or micelles. The vesicles can rupture upon contact with the substrate in order to form a membrane. 
         [0028]    The surface can be a solid substrate. The substrate can include glass. The substrate can bear a pattern. The pattern can have different chemical or physical properties than non-patterned regions of the substrate. The substrate can include one or more selected from the group consisting: metal, plastic, rubber, and silicone. 
         [0029]    The substrate can include a liquid. The substrate can be more viscous than the confining liquid. The substrate can be a gel. The substrate can be a hydrogel. 
         [0030]    The method can further include translating the fluidic device about the substrate while dispensing the one or more surfactant-containing liquids. 
         [0031]    The method can further include dispensing one or more processing solutions. The one or more processing solutions can comprise a chemical conjugation agent addressing a functional group in the membrane. The one or more processing solutions can comprise a membrane-soluble dye. The one or more processing solutions can comprise an antibody. The one or more processing solutions can comprise a detergent. 
         [0032]    The processing solution can be dispensed in a sequential manner relative to the one or more surfactant-containing liquids. The processing solution can be dispensed simultaneously with the one or more surfactant-containing liquids. The surfactant-containing liquids can be dispensed using pulse-width modulation so that the membrane will have a variable composition. 
         [0033]    The method can further comprise removing a portion of the membrane. 
         [0034]    The membrane can be a surfactant multilayer membrane, a surfactant single layer membrane, a double lipid bilayer membrane, a single lipid bilayer membrane, or a lipid monolayer membrane. 
         [0035]    The method can further include depositing one or more additional components into the membrane. 
         [0036]    The one or more additional components can include one or more selected from the group consisting of: a protein, a nanoparticle, a microsphere, a virus particle, a vesicle, a cell, a bacterial cell, a surfactant molecule, a lipid molecule, and a non-lipid molecule. 
         [0037]    The method can further include modifying a local region of the membrane. The modifying step can include one or more selected from the group consisting of: an additive reaction, a cleavage reaction, and a dissociation reaction. 
       DEFINITIONS 
       [0038]    Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale &amp; Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. 
         [0039]    In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. 
         [0040]    The term “microfabrication” is meant to refer to a set of techniques used for fabrication of micro- or nanostructures. In certain preferred embodiments, microfabrication includes, but is not limited only to, the following techniques: photolithography, electron beam lithography, laser ablation, direct optical writing, thin film deposition (spin-coating, spray coating, chemical vapor deposition, physical vapor deposition, sputtering), thin film removal (development, dry etching, wet etching), replica molding (soft lithography), embossing, forming or bonding. 
         [0041]    The term “microchannel” is meant to refer to a tube with nano- or microscopic cross-section. In certain preferred embodiments, a microchannel or channel has a size in the range of 0.1-200 μm. In other preferred embodiments of the present invention, microchannels are fabricated into microfluidic devices by means of microfabrication. 
         [0042]    The term “macrochannel” is meant to refer to a tube of size larger than a microchannel (&gt;200 μm) 
         [0043]    The term “channel” is meant to refer to either a microchannel or a macrochannel. 
         [0044]    The term “fluidic device” is meant to refer to a device which is used to handle and move fluids. A microfluidic device is a fluidic device. A capillary is a fluidic device. 
         [0045]    The term “microfluidic device” is meant to refer to the microfabricated device comprising microchannels or circuits of microchannels, which are used to handle and move fluids. Preferably, microfluidic devices may include components like junctions, reservoirs, valves, pumps, mixers, filters, chromatographic columns, electrodes, waveguides, sensors, etc. Microfluidic devices can be made of polymer (e.g., PDMS, PMMA, PTFE, PE, epoxy resins, thermosetting polymers), amorphous (e.g., glass), crystalline (e.g., silicon, silicon dioxide) or metallic (e.g., Al, Cu, Au, Ag, alloys) materials. In certain preferred embodiments, a microfluidic device may contain composite materials or may be a composite material. The microfluidic pipette is a microfluidic device. 
         [0046]    The term “membrane” is meant to refer to a molecular film with two-dimensional fluidity. 
         [0047]    The term “object of interest” is meant to refer to the material entity to be studied, investigated, transported, positioned, separated, or otherwise influenced or modified by means of the invention. 
         [0048]    The term “membrane attached” is meant to refer to a relationship between the membrane and the object of interest, which is characterized by a covalent or non-covalent binding or anchoring interaction. 
         [0049]    The term “membrane embedded” is meant to refer to a relationship between the membrane and the object of interest, which is characterized by the object of interest being located within the physical boundaries of the membrane. 
         [0050]    The term “reservoir” is meant to refer to the liquid volume that encapsulates the membrane. 
         [0051]    The term “channel exit” is meant to refer to an open end of a channel that leads into the open volume. 
         [0052]    The term “flow-recirculating fluidic device” is meant to refer to a fluidic device that features outflow from the device and aspiration back into the device, such that fluid leaving the device is fully or partially returned into the device. An example of a flow-recirculating device includes two closely-spaced capillaries. 
         [0053]    The term “flow-recirculating microfluidic device” is meant to refer to a microfluidic device that features outflow from the device and aspiration back into device, such that fluid leaving the device is fully or partially returned into the device. 
         [0054]    The term “mobility” is meant to refer to a parameter of the object relating its velocity to applied force. 
         [0055]    The term “processing solution” is meant to refer to a solution which is delivered by the flow-recirculation device to the surface. Examples of functions of processing solution are membrane fabrication, removal, and functionalization. 
         [0056]    The term “membrane source” is meant to refer to the processing solution used to fabricate the membrane. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0057]    Artificially created lipid membranes are versatile structures for mimicking biomembranes. Lipid membranes can be formed on solid supports, leading to enhanced stability of the molecular film. This enables functional studies of membrane properties, membrane-associated molecules and membrane proteins, and aids the development of applications such as biosensing, 2D chemical reactions and catalysis. 
           [0058]    Aspects of this disclosure describes a method to fabricate, modify, remove and utilize a two-dimensional fluid membrane (later referred to as “membrane”) on a surface. The key aspect of the method is the localized assembly of a mesoscale membrane from precursors on a surface and its subsequent manipulation, using an open volume microfluidic device for lipid delivery. This membrane assembly and manipulation method is especially favorable for the convenient and reproducible preparation of molecular films of desired composition and size on a solid support. Compared to other supported membrane preparation methods, this method is more versatile and reproducible. 
           [0059]      FIGS. 1A and 1B  depict a side view and a top view, respectively, of an exemplary embodiment of the fabrication of a 2D fluidic film by means of a flow-recirculating fluidic device comprising a surface  0101 , covered by a liquid  0102 . The device comprises three channels  0103 ,  0104  capable of producing a hydrodynamically confined flow (“HCF”)  0105  near the surface, while the channels can be translated  0106  relative to the surface. A processing solution, delivered though the channels and confined hydrodynamically, contains membrane precursor (“membrane source”), which when brought into contact with the surface can form a two-dimensional fluid membrane (“membrane”)  0107 . 
           [0060]      FIG. 2A  shows exemplary multiplexing  0203  of two processing solutions ( 0201 ,  0202 ) into the hydrodynamically confined flow  0204  sequentially.  FIG. 2B  shows an embodiment in which the flow-recirculating fluidic device allows two ( 0201 ,  0202 ) or more processing solutions to be applied in the hydrodynamically confined flow simultaneously ( 0205 ) through one or more channels. 
           [0061]      FIG. 3A  illustrates an exemplary embodiment in which two or more membrane sources are applied sequentially to fabricate a series of compositionally different membranes ( 0301 ,  0302 ), when brought into contact with the surface ( 0303 ). Each spot is fabricated from one individual membrane source. 
           [0062]      FIG. 3B  illustrates an exemplary embodiment in which multiplexing of different membrane sources is used to fabricate membranes of variable composition. Pulse width modulation ( 0307 ) is used for variable membrane composition in each individual spot ( 0306 ). 
           [0063]      FIG. 3C  illustrates an exemplary embodiment in which coupling of multiplexing of membrane sources to translation ( 0309 ) of the surface relative to the channels is used to fabricate a spatially heterogeneous membrane geometry ( 0308 ). 
           [0064]      FIGS. 4A and 4B  illustrate an exemplary embodiment in which one or more of the processing solutions ( 0402 ) can be used to remove ( 0405 ) the membrane locally ( 0403 ).  FIG. 4A  shows an exemplary membrane geometry before removal.  FIG. 4B  shows two new, disconnected membrane geometries after removal. 
           [0065]      FIGS. 5A and 5B  illustrate an exemplary embodiment in which a hydrodynamically confined processing solution ( 0502 ) modifies a membrane locally ( 0505 ).  FIG. 5A  shows an exemplary membrane geometry before local modification.  FIG. 5B  shows the same membrane geometry after local modification. 
           [0066]      FIGS. 6A and 6B  illustrate an exemplary embodiment in which a fabricated membrane is either immobile on the surface ( FIG. 6A ) or grows laterally by membrane spreading ( FIG. 6B ). 
           [0067]      FIGS. 7A-7E  illustrate an exemplary embodiment in which a surface ( 0701 ) is covered with a pattern ( 0702 ) ( FIG. 7A ).  FIG. 7A  shows an exemplary patterned surface with different properties in different patterns, wherein the surface has either the same properties over the entire pattern ( 0703 ) or has continuously variable properties ( 0704 ). In one embodiment ( FIG. 7C ) the membrane can be deposited from the membrane source ( 0705 ) selectively ( 0706 ) onto the pattern ( 0702 ). In one exemplary embodiment ( FIG. 7D ), some membrane properties (e.g., diffusivity) are different inside ( 0709 ) and outside ( 0708 ,  0709 ) the pattern. In one exemplary embodiment ( FIG. 7E ), the membrane can selectively propagate ( 0710 ) on the pattern. 
           [0068]      FIGS. 8A-8I  illustrate an exemplary embodiment in which an field or gradient ( 0803 ) is applied along some parts of the membrane ( 0802 ) on the surface ( 0801 ) ( FIG. 8A ), the field or gradient causes membrane propagation ( 0804 ) ( FIG. 8B ), propagation of membrane-attached molecules or particles ( 0805 ) ( FIG. 8C ), spatial separation of molecules or particles ( 0806 ,  0807 ) ( FIG. 8D ). The source of the field ( 0808 ) can be located in or on the surface ( FIG. 8E ), or above or below the surface ( FIG. 8F ), and can be located in an external device ( 0809 ), e.g., the hydrodynamically confined fluidic device. The field or gradient can be homogenous or non-homogenous ( 0810 ) ( FIG. 8G ). In one embodiment, the field or gradient changes polarity or direction. The point of polarity or direction change ( 0812 ) can be a stable stagnation point for membrane components or membrane attached components ( 0811 ), which are migrating in the field or gradient ( FIG. 8H ). The stagnation point can be moved across the surface ( 0813 ) ( FIG. 8I ), and used for separation, or concentration of membrane components or membrane attached components. 
           [0069]      FIGS. 9A-9C  illustrate the examples of types of membranes which can be fabricated, multiple layers ( 0902 ) ( FIG. 9A ), bilayers ( 0903 ) ( FIG. 9B ), or monolayers ( 0904 ) ( FIG. 9C ). 
           [0070]      FIGS. 10A and 10B  shows examples of membrane fabrication mechanisms. Exemplary embodiments are rupture ( 1003 ) of vesicles ( 1002 ), which form a membrane ( 1004 ) on the surface ( 1001 ) ( FIG. 10A ), and fusion of vesicles ( 1006 ) into an existing membrane ( 1005 ) on the surface ( 1001 ) ( FIG. 10B ). 
           [0071]      FIGS. 11A and 11B  illustrate the utilization of a flow-recirculating microfluidic device ( 1101 ) for the fabrication of a membrane ( 1104 ) on a surface ( 1102 ). The device comprises inflow ( 1106 ) and outflow ( 1105 ) channels, which generate a flow recirculation volume ( 1103 ). The inflow is selected through a valveless switching chamber ( 1107 ), comprising a number of supply ( 1108 ) and vacuum ( 1110 ) channels.  FIG. 11A  is a schematic top down view,  FIG. 11B  a perspective view, where distance to the surface h and device positioning angle α are marked. 
           [0072]      FIGS. 12A-B  show fluorescence micrographs of a fabricated nonspreading lipid membrane patch ( 1202 ) immediately after deposition (A) and after three minutes (B). The channel positions are marked by white rectangles ( 1201 ) in (A), and the initial patch perimeter (t=0 min,  1203 ) by a white circle in (B).  FIG. 12C  shows the development of the fluorescence intensity of the membrane attached label over time. The inset shows the uniformity of the membrane spot along a surface coordinate. 
           [0073]      FIGS. 12D-E  show fluorescence micrographs of a fabricated spreading lipid membrane patch ( 1204 ) immediately after deposition (D) and after eight minutes ( 1204 ) (E). The channel positions are marked by white rectangles ( 1201 ) in (D), and the initial patch perimeter (t=0 min,  1203 ) by a white circle in (E).  FIG. 12F  shows the radius increase of the patch over time. 
           [0074]      FIGS. 12G-H  show fabrication of a series of membrane patches ( 1205 ) of systematically changing composition, achieved by applying pulse width modulation flow switching between two processing solutions containing different membrane sources. 
           [0075]      FIG. 12G  shows the fluorescence micrographs of the membrane component originating from source  1 , and  FIG. 12H  shows the fluorescence micrographs of the membrane component originating from source  2 .  FIG. 12I  shows the quantification of the fluorescence with respect to the PWM ratio of the two components ( 1206 ,  1207 ). 
           [0076]      FIG. 12J  shows the fusion of a continuously applied second membrane source ( 1209 ) with an already fabricated membrane ( 1208 ). Both membrane and source were supplied by a flow-recirculating microfluidic device. The prefabricated membrane was stained with one fluorescent label, and the second membrane source with another. Both fluorescence channels are depicted. The original membrane patch grows due to incorporation of the membrane material from the second source ( 1210 ). 
           [0077]      FIGS. 13A-C  show fluorescence micrographs of exemplary membrane geometries, fabricated by translation of the flow-recirculating microfluidic device relative to the surface during fabrication. 
           [0078]      FIG. 13D  shows the fabrication of two partially overlaid membranes ( 1302  and  1303 ) immediately after fabrication, and at t=22 min. Each membrane is stained with a different fluorescent label. Both membranes mix by diffusion. 
           [0079]      FIG. 13E  shows the fluorescence intensity development over time for both dyes along a horizontal surface coordinate. Solid and dashed lines represent each of the two fluorescent labels, respectively. 
           [0080]      FIGS. 14A-14E  are micrographs of localized membrane removal from prefabricated membrane lanes ( 1401 ), using a processing liquid containing a detergent ( 1402 ). The formed gap ( 1403 ) on the lower lane is repaired by fabricating membrane from another membrane source ( 1404 ). Diffusion across the repaired gap confirms fluidic connectivity ( 1405 ). 
           [0081]      FIGS. 15A-15D  are fluorescence micrographs of the stepwise functionalization of a fabricated membrane ( 1501 ). A fluorescently-labeled primary antibody ( 1502 ) against a membrane constituent (biotin) is applied first, and a fluorescently-labeled secondary antibody ( 1503 ) against the first antibody is applied second, both from different processing solutions from the flow-recirculating microfluidic device. The top row shows a time series of the fluorescence of a membrane-attached fluorescent dye, the middle row a time series of the fluorescence of the primary antibody, and the bottom row a time series of the fluorescence of the secondary antibody. 
           [0082]      FIGS. 16A-16D  are micrographs of a hydrodynamic trapping experiment. A spreading membrane ( 1601 ) is fabricated first, and a non-spreading membrane ( 1602 ) second ( FIG. 16A ). Outflow from the center channel of the flow-recirculating microfluidic device ( 1603 ) is reversed ( FIG. 16B ), leading to accumulation of membrane material ( 1604 ) from the non-spreading material in the stagnation point of the flow field. 
           [0083]      FIGS. 17A-17C  are fluorescence micrographs of directed migration ( 1704 ) of a spreading membrane ( 1701 ) inside specific patterns ( 1702 ) on a patterned surface ( 1703 ). 
           [0084]      FIGS. 18A-18C  are a time series of three fluorescence micrographs of trapping and concentration control of fluorescent nanoparticles  1801  at different trapping conditions defined by the inflow rate of the flow-recirculating microfluidic device. Immobile particles ( 1802 ) are unaffected by the flow. 
           [0085]      FIGS. 19A and 19B  show two exemplary modes of transport of a membrane-attached object  1905  by moving the stagnation point. The object is trapped in the stable stagnation point  1904  created by the field source  1903  in the reservoir  0702 .  FIG. 19A  shows transport by positioning the stagnation point by translation of the field source along vector  1906  parallel to the membrane plane.  FIG. 19B  shows the transport by positioning the stagnation point by changing the ratio of the field strengths of the fields generated by the sources  1903 . 
           [0086]      FIGS. 20A-20C  depicts separation of membrane-attached objects  2006  and  2007  by scanning motion  2005  of field source  2003  parallel to membrane  2001  inside liquid volume  2002 . The field  2004  is used to create a stagnation point  2009 . If the stagnation point is translated by scanning motion with velocity ν, the membrane-attached objects experience a force  2013  depending on their distance from the stagnation point. The force is balanced by the viscous drag  2012 , which determines whether the membrane-attached objects are able to follow the scanning motion  2006 , or stay behind  2007 .  FIG. 20B  shows the force on the objects depending on the position relative to the stagnation point ( 2009 ).  FIG. 20C  shows the balance between the drag ( 2013 ) on the membrane-attached object ( 2011 ), which is produced by the field ( 2004 ) and the viscous drag produced by the membrane ( 2012 ). 
           [0087]      FIGS. 21A-21E  show exemplary uses of the invention.  FIG. 21A  depicts the process of bringing two membrane-attached objects  2102  and  2103  together in order to bind them to each other  2105 .  FIG. 21B  depicts the full or partial removal  2108  of a membrane-attached object  2106  from the membrane by means of a chemical reaction initiated by delivery of a reagent  2107 .  FIG. 21C  depicts the morphological change of a membrane-attached object  2109  initiated by delivery of a reagent  2110 .  FIG. 21D  shows the formation of a gel  2113  from precursor  2112  for fixation of membrane-attached objects in the membrane.  FIG. 21E  shows an example of the arrangement of membrane proteins into an ordered two-dimensional assembly. 
           [0088]      FIGS. 22A-22F  depict heat-assisted separation or fixation of membrane-attached objects, wherein the mobility of the objects is dependent on the temperature.  FIGS. 22A and 22B  show an exemplary setup where the heating zone  2202  is co-localized with stagnation point  2203 . Mobility and diffusion of the objects  2207  outside of the heated-zone  2208  is reduced.  FIG. 22C  depicts the co-localization of an electromagnetic radiation guide  2209  with aspiration channel  2203 , creating a heated zone  2210  around the stagnation point. In an exemplary embodiment, the guide is an optical fiber.  FIG. 22D  depicts an optical heating by means of focusing radiation with an objective  2211 .  FIG. 22E  depicts heating by convection from a fluid stream with an elevated temperature  2213 .  FIG. 22F  depicts heating by a substrate-embedded heater  2214 . In one embodiment, the substrate-embedded heater can be a resistive heater. 
           [0089]      FIG. 23  shows exemplary use of the invention to deposit lipid monolayer films on patterned polymer surface. Element  2301  is the unexposed TEFLON® AF coated deposition area. Element  2302  is the e-beam exposed TEFLON® AF coated area. Element  2303  is the zone of the lipid membrane source delivery. Element  2304  is the flow-recirculating device tip. Element  2305  is the one of several alignment marks and numerals. 
       
    
    
     DETAILED DESCRIPTION 
       [0090]    This disclosure describes a method and a system to fabricate, modify, remove and utilize a two-dimensional fluid membrane on a surface.  FIGS. 1A and 1B  depict an embodiment of the method and system. 
         [0091]    The invention comprises a surface  0101  covered by a liquid  0102 , and two or more channels  0103 ,  0104 , which are part of a “fluidic device” that is capable of producing a hydrodynamically confined flow  0105  near the surface  01010 . The channels of the device can be translated relative to the surface  0101  along or more axes  0106  such that the hydrodynamically-confined flow exposes desired areas of the surface  01010 . The system also includes one or more processing solutions, delivered though one or more of the channels to the surface, and confined hydrodynamically in a volume between the outlet of the device and the surface. One or more of the processing solutions contain a “membrane source”, which when brought into contact with the surface forms a two-dimensional fluid membrane (“membrane”)  0107 . The membrane  0107  has two-dimensional fluidity, meaning that the building blocks of the membrane  0107  as well as the object attached to it can freely migrate within the membrane  0107 . 
         [0092]    Two or more processing solutions ( 0201 ,  0202 ) are supplied to the flow-recirculating device ( FIG. 2 ). The device comprises a switching device  0203  as depicted in  FIG. 2A  that enables selection of an active processing solution, which is recirculated in the recirculation zone  0204 . The switching device  0203  can comprise a pneumatic or electric valve or can be valveless. Valveless switching can include flow steering. The device can include a multicomponent recirculation zone  0205 , where individual flows  0201  &amp;  0202  are combined as co-flows ( FIG. 2B ). The flow rates in the device can be changed, which influences the size and geometry of the recirculation zone, as well as the confinement. 
         [0093]    One embodiment of a flow-recirculating fluidic device is the multifunctional pipette described in International Publication Nos. WO 2011/067670 and WO 2012/153192 and Ainla, et al., Lab Chip 2012, DOI: 10.1039/C2LC20906C). This device allows valveless switching of up to 4 different processing solutions, which are provided from reservoirs inside the device. Other embodiments can comprise metal, glass or plastic capillaries, which are fabricated so that they can be brought sufficiently close to the solid surface and can be brought or are sufficiently close to each other. 
         [0094]    External or internal reservoirs can be used for storage and delivery of processing solutions. In one embodiment, the channels are in the size range of 20 to 40 μm, positioned between 1-50 μm above the surface, and 5-50 μm separated from each other. The invention can comprise larger channels, wherein the channel separation scales with channel size. In one embodiment, the flow of processing solution through the recirculation zone can be driven by pressure and vacuum, or by electrical fields. 
         [0095]    One or more processing solutions contain a membrane source, which in a preferred embodiment comprises small unilamellar vesicles. Other types of surfactant assemblies can also serve as membrane source. Nonlimiting examples of membrane sources are liposomes, phospholiposomes and niosomes. One or more membrane sources are simultaneously or sequentially recirculated by the flow-recirculating fluidic device, such that they are brought in contact with the surface. Upon contact, the membrane source adheres to the surface, and is there transformed into a membrane. H. Schonherr, J. M. Johnson, P. Lenz, C. W. Frank, S. G. Boxer, Vesicle adsorption and lipid bilayer formation on glass studied by atomic force microscopy, Langmuir 20 (2004) 11600-11606. Different types of membrane can be fabricated. In one embodiment, the fabricated membrane is a monolayer ( 0904 ) ( FIG. 9C ). In another embodiment, the fabricated membrane is a double layer (or bilayer) ( 0903 ) ( FIG. 9B ). In another embodiment, the fabricated membrane is a multilayer ( 0902 ) ( FIG. 9A ). 
         [0096]      FIG. 10A-B  shows examples of possible mechanisms of transformation of the membrane source into the membrane. Exemplary embodiments are rupture ( 1003 ) of vesicles ( 1002 ), which form a membrane ( 1004 ) on the surface ( 1001 ) ( FIG. 10A ), and fusion of vesicles ( 1006 ) into an existing membrane ( 1005 ) on the surface ( 1001 ) ( FIG. 10B ). Nonlimiting examples of lipid sources are small unilamellar vesicles fabricated from are 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Soy L-α-phosphatidylcholine (PC), and 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP), and vesicles containing membrane fractions obtained from biological cells or cell components. Examples of surfaces are soda-lime glass, borosilicate glass, quartz, and oxidized silicon (silicon dioxide). The transformation of the membrane source to a membrane can occur in different ways. 
         [0097]    The time of exposure of the surface by the membrane source determines the coverage of the surface, and the fluidic properties of the formed membrane. Short exposure times prevent the formation of a coherent membrane that consistently covers the entire exposed surface. If the surface is insufficiently covered by the membrane source, the membrane is not coherent over the exposed area, and transport within the membrane is not possible. In one embodiment, the time of exposure of the surface is chosen to allow sufficient coverage for two-dimensional transport. In another embodiment, the time of exposure is chosen to provide insufficient coverage for two-dimensional transport. 
         [0098]    In one example, membrane source is deposited onto the surface such that the surface area exposed by the flow recirculation is fully covered. In this case deposition stops upon full coverage of the exposed surface area (“nonspreading deposition”) ( FIG. 6A ,  10 A). In a different example, membrane source is deposited onto the surface in the same way, but after complete coverage, membrane source continues to be deposited by fusing with the existing membrane, and the membrane area on the surface increases by continuous spreading (“spreading deposition”) ( FIG. 6B ,  10 B). 
         [0099]    An example of nonspreading deposition is shown in  FIG. 12A-C , using POPC vesicles as membrane source. Details are provided in the example section (Example 1). An example of spreading deposition is shown in  FIG. 12D-F , using DOTAP vesicles as membrane source. Details are provided in the example section (Example 2). In one embodiment the membrane components are fully or partially exchanged while membrane material is supplied by the hydrodynamic flow confinement. If the membrane and the vesicles from the membrane source are in close contact, individual membrane components can be transferred from either the membrane to the source or vice versa. One example is the transfer of membrane proteins; another example is the enrichment of the membrane with cholesterol; still another example is the exchange of lipid molecules. 
         [0100]    Different membrane sources are either simultaneously, or sequentially supplied. By this means a membrane of desired composition can be fabricated. One example of simultaneous application is on-device mixing prior to supplying the membrane sources to the flow recirculation, another example is mixing on the surface after individual membrane sources have been supplied to the recirculation zone. One example of sequential application is pulse-width-modulation-like flow switching inside the flow-recirculating fluidic device. Another example of sequential application is the insertion of membrane source into a membrane by means of “spreading deposition”. 
         [0101]    In one embodiment, multiple individual membrane deposits of well-defined and individually different composition can be fabricated on selected regions on a surface, using a different membrane source on each selected region. Here, the composition of the membrane source is defined prior to deposition.  FIG. 3A  illustrates an exemplary embodiment in which two or more membrane sources are applied sequentially to fabricate a series of compositionally different membranes ( 0301 ,  0302 ), when brought into contact with the surface ( 0303 ). Each spot is fabricated ( 0305 ) from one individual membrane source, coming from the flow-recirculating fluidic device  0304 . In another embodiment, the composition of the membrane source is defined during deposition. Switching between different membrane sources, or multiplexing, is used to create such membrane spots of variable composition. In a preferred embodiment, pulse width modulation is used for multiplexing two or more different membrane sources in order to define the membrane composition.  FIG. 3B  illustrates an exemplary embodiment in which multiplexing of different membrane sources is used to fabricate membrane regions of variable composition. Pulse width modulation ( 0307 ) is used for variable membrane composition in each individual spot ( 0306 ). 
         [0102]    In the example section (Example 3), sequential deposition (multiplexing) of two differently fluorescently labeled POPC membrane sources is demonstrated.  FIGS. 12G-H  show fabrication of a series of membrane patches ( 1205 ) of systematically-changing composition, achieved by applying pulse width modulation flow switching between two processing solutions containing different membrane sources.  FIG. 12G  shows the fluorescence micrographs of the membrane component originating from source  1 , and  FIG. 12H  shows the fluorescence micrographs of the membrane component originating from source  2 .  FIG. 12I  shows the quantification of the fluorescence with respect to the PWM ratio of the two components ( 1206 ,  1207 ).  FIG. 12J  (Example 4) shows the fusion of a continuously applied second membrane source ( 1209 ) with an already fabricated membrane ( 1208 ) (“spreading deposition”). Both membrane and source were in this example supplied by a flow-recirculating microfluidic device. Both fluorescence channels are depicted. The original membrane patch grows due to incorporation of the membrane material from the second source ( 1210 ). 
         [0103]    In one embodiment, membranes can be deposited in different geometries. If the surface is translated relative to the flow-recirculating fluidic device, extended areas on the surface can be covered with the membrane. The shape and geometry of the membrane area deposited depends on the trajectory, speed, and sequence of the translations. In one embodiment, the time of deposition is chosen such that the membrane is coherently covering the whole deposition area and has two-dimensional fluidity over the entire deposition area. 
         [0104]    In one embodiment, the invention comprises a positioning device that allows translation of the device relative to the surface. Examples of positioning devices are micromanipulators and scanning stages. In a preferred embodiment, the positioning device features electronic control. In another preferred embodiment the setup comprises a control unit, which allows defining the speed and trajectory of the translation. 
         [0105]    In another preferred embodiment, the control unit can also determine the flow rates in one or more channel(s). In one embodiment, the composition of the membrane is the same over the entire deposited membrane geometry. In another embodiment, the composition of the membrane differs over the entire deposited membrane geometry, creating a spatially heterogeneous geometry with respect to membrane composition. 
         [0106]    In one embodiment, different deposited geometries are overlapping and are fluidically connected ( 0308 ).  FIG. 3C  illustrates an exemplary embodiment in which coupling of multiplexing of membrane sources to translation ( 0309 ) of the surface relative to the channels is used to fabricate a spatially heterogeneous membrane geometry ( 0308 ). In another embodiment, different geometries different deposited geometries are not overlapping, and are not fluidically connected ( FIG. 3B ). In the example section (Example 5), exemplary membrane geometries, fabricated by translation of the flow-recirculating microfluidic device relative to the surface during fabrication are described.  FIG. 13A-C  show fluorescence micrographs of some written geometries. 
         [0107]    In one embodiment, membranes of different composition are deposited in overlapping geometries in such a way, that the geometries are fluidically connected. Membrane components can cross over from one geometry to the other by means of diffusion. Example 6 demonstrates the fabrication and diffusional exchange between overlapping membrane geometries.  FIG. 13D  shows the fabrication of two partially overlaid membranes ( 1302  and  1303 ) immediately after fabrication, and after ˜20 min. Each membrane is stained with a different fluorescent label. Both membranes mix by diffusion. Examples of uses for such diffusionally coupled membrane geometries are two-dimensional reaction systems, devices for membrane protein analysis, sensors and mimics of biological intercellular transport functions. The ability to generate compositional gradients in a deposited membrane geometry allows for the creation of driving forces for transport, and self-assembly, as well as concentration control of membrane components. A specific application area is the generation of functional self-assembled films on or within the deposited membrane, for example in the areas of photonics, catalysis or chemical transformations. 
         [0108]    In one embodiment, a chemical reactant that dissolves or decomposes the deposited membrane is recirculated on a selected area on deposited membrane geometry, such that the deposited membrane is disassembled and removed from the surface in the exposed area. In another embodiment, new membrane is deposited onto the surface from which the membrane was removed, re-connecting the separated membrane geometries. In another embodiment, new membrane is deposited onto the surface from which the membrane was removed, connecting either geometry with a different membrane geometry in the vicinity. This allows the separation, repair and reconfiguration of deposited membrane geometries, as well as the establishment of reconfigurable membrane networks. In another embodiment, the membrane material that was removed from the surface is collected in an external or on-chip reservoir and used for membrane post-processing or chemical analysis.  FIGS. 4A and 4B  illustrate an exemplary embodiment in which one or more of the processing solutions ( 0402 ) can be used to remove ( 0405 ) the membrane locally ( 0403 ).  FIG. 4A  shows an exemplary membrane geometry before removal.  FIG. 4A  shows two new, disconnected membrane geometries after removal. Example 7 demonstrates localized membrane removal from prefabricated membrane lanes ( 1401 ), using a processing liquid containing a detergent ( 1402 ). The formed gap ( 1403 ) on the lower lane is repaired by fabricating membrane from another membrane source ( 1404 ). Diffusion across the repaired gap confirms fluidic connectivity ( 1405 ).  FIGS. 14A-14E  provide micrographs of this example. 
         [0109]    In one embodiment, a deposited membrane geometry is functionalized with a chemical or biological reagent. 
         [0110]    In one embodiment, the modification involves an additive reaction, where a reagent is coupled to the membrane. In one embodiment, the modification involves a cleavage reaction, or dissociation.  FIG. 21B  depicts the full or partial removal  2108  of a membrane-attached object  2106  from the membrane by means of a chemical reaction initiated by delivery of a reagent  2107 . In one embodiment the reaction alters or breaks covalent or coordination bonds. In one embodiment, the reaction involves non-covalent binding. In one embodiment, the non-covalent binding is based on either nucleic acid hybridization, ligand-receptor affinity or antigen-antibody recognition. In one embodiment, the two or more components of the membrane are reacting with each other or binding to each other. 
         [0111]    Examples of chemical or biological reagents are proteins, peptides, sugars, lipids, DNA, enzymes, ions, ligands, and small organic molecules. In one embodiment, global modification of a deposited membrane is performed by adding the reagent to the liquid. In another embodiment, modification is performed using a conventional fluidic device such as a glass needle. In another embodiment, local modification is performed by means of the processing solutions of the flow-recirculating fluidic device. In one aspect, the processing solution contains a chemical conjugation agent addressing a functional group available in the membrane. 
         [0112]    In one aspect, the processing solution contains a functional molecule coupled to a chemical conjugation agent addressing a functional group available in the membrane. 
         [0113]    In another aspect, the processing solution contains a functional molecule addressing a chemical conjugation group available in the membrane. In another aspect, the processing solution contains a functional molecule addressing a complexing group available in the membrane. In another aspect, the processing solution contains a functional molecule addressing a receptor available in the membrane. In another aspect, the processing solution contains a functional molecule addressing a ligand available in the membrane. In one embodiment, the processing solution contains a membrane soluble dye. In one embodiment, the processing solution contains an antibody. The processing solution can contain a detergent. In another aspect, the processing solution causes morphological change of the membrane attached objects ( FIG. 21C ).  FIG. 21C  depicts the morphological change of a membrane-attached object  2109  initiated by delivery of a reagent  2110 . 
         [0114]    In one aspect, the processing solution causes fixation or immobilization of the membrane or membrane components. In one aspect, the fixation is caused by antibody binding. In one aspect, the fixation is caused by receptor-ligand binding. In one aspect, the fixation is caused by gel formation near the membrane ( FIG. 21D ).  FIG. 21D  shows the formation of a gel  2113  from precursor  2112  for fixation of membrane-attached objects in the membrane. In one aspect, the gel is used to remove the membrane from the supporting substrate. In one embodiment, the chemical treatment of membrane or membrane-attached objects is used simultaneously or subsequently with concentration or separation. 
         [0115]      FIGS. 5A and 5B  illustrate an exemplary embodiment in which a hydrodynamically confined processing solution ( 0502 ) modifies a membrane locally ( 0505 ).  FIG. 5A  shows an exemplary membrane geometry before local modification.  FIG. 5B  shows the same membrane geometry after local modification. 
         [0116]    Membrane functionalization can be used in combination with the diverse functionalities of a microfluidic device. In one embodiment, the flow-recirculating fluidic device is a microfluidic device. In another embodiment, the flow-recirculating fluidic device is connected to a microfluidic device. Examples of microfluidic functionalities are the delivery of single chemical or biochemical solutions to the membrane or membrane-attached object, the processing and subsequent delivery of multiple chemical or biochemical solutions to the membrane or membrane-attached object, where processing comprises mixing, dilution, switching and temperature regulation. 
         [0117]    Further examples of microfluidic functionalities are the processing of aspirated fluid. The aspirated fluid can comprise membrane, membrane components, membrane-attached objects, and fragments or products of chemical reactions involving membrane or membrane-attached objects. Examples of aspirated fluid comprise DNA, proteins, peptides, lipids, sugars, ions, and ligands. Microfluidic processing functionalities for aspirated fluid can comprise sensing, partitioning, division into aliquots, concentration, dilution, chemical modification, digestion, fractionation, separation, and detection. In some embodiments, the aspirated fluid can be transferred to external processing devices. 
         [0118]    Example 8 demonstrates sequential membrane functionalization.  FIGS. 15A-15D  provide fluorescence micrographs of the stepwise functionalization of a fabricated membrane ( 1501 ). A fluorescently-labeled primary antibody ( 1502 ) against a membrane constituent (biotin) is applied first, and a fluorescently-labeled secondary antibody ( 1503 ) against the first antibody is applied second, both from different processing solutions from the flow-recirculating microfluidic device. The top row shows a time series of the fluorescence of a membrane-attached fluorescent dye, the middle row a time series of the fluorescence of the primary antibody, and the bottom row a time series of the fluorescence of the secondary antibody. 
         [0119]    In one embodiment, the membranes are deposited on a patterned surface. The feature size of a pattern can be between 10 nm and the size scale of the entire surface. Patterns can be regions that are physically or chemically different from the remainder of the surface. 
         [0120]    Chemical patterns can have a different surface chemistry or different material than the remainder of the surface. Physical patterns can have differences in surface morphology, such as roughness. Examples of chemical patterns are protein coatings, photoresist or polymer coatings, hydrogel coatings, self-assembled monolayers, or deposited thin films. Examples of physical patterns are regular or irregular arrays of particles or pillars, surface roughness resulting from polishing, etching, or sputtering. Examples of methods to produce patterns on surfaces are localized treatment with chemicals, lithography, plasma treatment, coating, physical and chemical deposition, wet and dry etching, chemical and physical etching. 
         [0121]    In another aspect, the patterns are defined by the geometry of the features. Examples of geometric patterns are patches, lanes, and interconnected combinations of patches and lanes of variable sizescales from 10 nm to the size scale of the surface. Examples of methods to define geometries are lithography, engraving, embossing, direct writing techniques, and physical masks. 
         [0122]    Patterns of different type and size scale can coexist on the same surface.  FIGS. 7A-7E  illustrates an exemplary embodiment in which a surface ( 0701 ) is covered with a geometric pattern ( 0702 ) ( FIG. 7A ).  FIG. 7A  shows an exemplary patterned surface with different properties in different patterns, wherein the surface has either the same properties over the entire pattern ( 0703 ) or has continuously variable properties ( 0704 ). Examples of functions of the pattern with respect to the membrane source are either differential adhesion properties of membrane material ( FIG. 7C ) or differential transport or partitioning properties ( FIG. 7D ) of the membrane material or of individual membrane components or of membrane-associated materials and objects. In one embodiment ( FIG. 7C ), the membrane can be deposited from the membrane source ( 0705 ) selectively ( 0706 ) onto the pattern ( 0702 ). In one exemplary embodiment ( FIG. 7E ), the membrane can selectively propagate ( 0710 ) on the pattern. 
         [0123]    Example 10 demonstrates controlled lipid flow in patterned 2D channels. In  FIGS. 17A-17C , fluorescence micrographs of directed migration ( 1704 ) of a spreading membrane, which is labeled with a fluorescent dye ( 1701 ), and deposited inside specific patterns ( 1702 ) on a patterned surface ( 1703 ) are shown. 
         [0124]    In one embodiment, a membrane can be exposed to a field or a gradient. The field or gradient acts on membrane or its components causing migration of membrane or membrane components or causing modification of the membrane. Examples of fields are hydrodynamic flow field, electrical field, magnetic fields, and electromagnetic fields. Examples of gradients are surface tension gradient, thermal gradient, solution composition gradient, and surface chemical gradient. The field or gradient can be applied during or after membrane deposition, or both. The source of the field ( 0808 ) or gradient can be part of the fluidic device, or can be part of the surface ( FIG. 8E ), or above ( FIG. 8F ) or below the surface, and can be located in an external device ( 0809 ), e.g., the hydrodynamically confined fluidic device. Sources of the fields or gradients, could be aspiration or injection flow channels, electrodes, permanent and electromagnets, optical fibers, focused light sources, boundaries of hydrodynamically confined flow, surface patterns, local chemical reactions, mechanical straining of substrate or membrane.  FIGS. 8A-8I  illustrate an exemplary embodiment in which an field or gradient ( 0803 ) is applied along some parts of the membrane ( 0802 ) on the surface ( 0801 ) ( FIG. 8A ). The field or gradient causes membrane propagation ( 0804 ) ( FIG. 8B ), propagation of membrane-attached molecules or particles ( 0805 ) ( FIG. 8C ), spatial separation of molecules or particles ( 0806 ,  0807 ) ( FIG. 8D ). The field or gradient can be homogenous or non-homogenous ( 0810 ) ( FIG. 8G ). 
         [0125]    In one embodiment, the field or gradient changes polarity or direction. In a preferred embodiment, the lateral component of the field or gradient changes polarity or direction. The point of polarity or direction change ( 0812 ) can be a stable stagnation point for membrane components or membrane attached components ( 0811 ), which are migrating in the field or gradient ( FIG. 8H ). The stagnation point can be moved across the surface ( 0813 ) ( FIG. 8I ), and used for separation, or concentration of membrane components or membrane attached components. 
         [0126]    In one embodiment, a field is applied that features a stagnation point in one or both of its lateral components. The stagnation point, in which the field changes polarity, is located in the area that is covered by the membrane. In one embodiment, the membrane components interact with the field, causing a stable stagnation point for these membrane components. In one embodiment, the stable stagnation point is used to concentrate or accumulate membrane components. In this embodiment, a static field source is translated across the membrane, translating the stable stagnation point. Membrane components or membrane-attached objects migrate in the plane of the membrane together with the stagnation point. 
         [0127]      FIGS. 19A and 19B  shows two exemplary modes of transport of a membrane-attached object  1905  by moving the stagnation point. The object is trapped in the stable stagnation point  1904  created by the field source  1903  in the reservoir  0702 .  FIG. 19A  shows transport by positioning the stagnation point by translation of the field source along vector  1906  parallel to the membrane plane.  FIG. 19B  shows the transport by positioning the stagnation point by changing the ratio of the field strengths of the fields generated by the sources  1903 . 
         [0128]      FIG. 20A  depicts separation of membrane-attached objects  2006  and  2007  by scanning motion  2005  of field source  2003  parallel to membrane  2001  inside liquid volume  2002 . The field  2004  is used to create a stagnation point  2009 . If the stagnation point is translated by scanning motion with velocity ν, the membrane-attached objects experience a force  2013  depending on their distance from the stagnation point. The force is balanced by the viscous drag  2012 , which determines whether the membrane-attached objects are able to follow the scanning motion  2006 , or stay behind  2007 .  FIG. 20B  shows the force on the objects depending on the position relative to the stagnation point ( 2009 ).  FIG. 20C  shows the balance between the drag ( 2013 ) on the membrane-attached object ( 2011 ), which is produced by the field ( 2004 ) and the viscous drag produced by the membrane ( 2012 ). 
         [0129]    The invention can be utilized to confine, trap, accumulate, position, move, transport, separate and extract objects which are attached to or embedded in a membrane. In one aspect, the invention comprises a method to control the local concentration of membrane attached objects. This control of the concentration means accumulation of the membrane attached objects in the vicinity of the stagnation point. 
         [0130]    To produce a stagnation point, a field is generated by a field source near the membrane. If projected to the membrane plane, then the field lines  2004  are converging towards a point (stagnation/sink point) near the field source  2003 . The field exerts a force, which is moving the objects towards the stagnation point. Since the objects are membrane-attached, they cannot follow the field towards the field source, and become stably trapped in the stagnation point. While trapped, the objects are governed by two opposing processes, 2D diffusion in the membrane, which would broaden their spatial distribution in the membrane, and field confinement, which is pulling them towards the stagnation point, and keeping them in it. This balance can be adjusted by changing the field strength, or force, around the stagnation point. By changing the field strength, or by moving the field source perpendicularly towards and away from the membrane, the force on the objects is adjusted. The adjustment allows control of the balance, and determines the concentration distribution of the membrane-attached objects around the stagnation point. The maximum achievable concentration has a limit, due to the crowding effects, which are more significant in 2D space than in 3D. 
         [0131]    By deliberately moving the stagnation point  2005 , the membrane-attached objects follow it, and are thus transported between desired areas, or regions, on the membrane. If the objects are located outside of the stagnation point, they experience a restoring force pulling them towards it. The magnitude of the force depends on the spatial offset of the object from the stagnation point. At small offsets, the force will increase with offset distance, which provides a negative feedback. However, the force has a maximum  2010 , after which it will drop rapidly  2008  with distance from the stagnation point  2009 . 
         [0132]    The force ( 2013 ) further depends on the field strength ( 2004 ) and size and shape of the membrane-attached object ( 2011 ). On the other hand, the transported object experiences viscous drag ( 2012 ), which is generated by the membrane and depends on the anchoring. The size, shape and anchoring contribute to a “mobility factor” (μ), which determines the relation (ν=μΦ) between transport velocity (ν) of the object and the field or gradient strength (Φ) around it. These are material properties of the membrane and the membrane-attached object. 
         [0133]    Positioning of the stagnation point can be achieved by scanning the field source or changing the field strength in two or more field sources ( FIGS. 7A-7E ). If the stagnation point ( 2009 ) is scanned ( 2005 ), the objects will experience a positional offset from the stagnation point ( FIG. 20B ). The larger the offset, the larger is the restoring force pulling the objects toward the stagnation point (negative feedback). However, this relation has maxima ( 2010 ) (σ max ), beyond which the restoring force will rapidly decrease ( 2008 ). If the scanning rate ν is chosen such that the force required to move the objects at this rate is less than the maximum (ν&lt;μΦ max ), the object will follow the stagnation point ( 2006 ). If the scanning rate is higher, the object will stay behind ( 2007 ). Since the restoring force will decrease rapidly after the maxima, it provides additional positive feedback for μ-based separation, with the limit μ limit =ν/Φ max . 
         [0134]    In some embodiments, the scanning rate or the force are varied during the separation to sequentially separate objects with different mobility factors μ from each other ( 2006 ,  2007 ). 
         [0135]    In some aspects, the invention comprises an additional means for local delivery of materials into the vicinity of the stagnation point. In some aspects, the material can be membrane material for formation of membrane ( FIG. 21B ). In another aspect, the material can alter the shape or size of the object and, therefore, would also alter the mobility factor (μ) ( FIGS. 21C-D ). The shape or size of the object can be altered by digestion ( FIG. 21C ) or conformation changes ( FIG. 21D ) or by formation of molecular assemblies in the membrane. 
         [0136]    In one aspect, the method is used in combination with a means for fixation of the membrane ( FIG. 21E ), which is beneficial to maintain positions of concentrated or separated objects. In some aspect the fixation is done by the local formation of a gel ( 2113 ). In some aspects, the gel  2113  can be used to remove membrane from the substrate for subsequent processing and analysis. 
         [0137]    Example 9 demonstrates hydrodynamic vesicle trapping.  FIGS. 16A-16D  provide micrographs of a hydrodynamic trapping experiment. A spreading membrane ( 1601 ) is fabricated first, and a non-spreading membrane ( 1602 ) second ( FIG. 16A ). Outflow from the center channel of the flow-recirculating microfluidic device ( 1603 ) is reversed ( FIG. 16B ), leading to accumulation of membrane material ( 1604 ) from the non-spreading material in the stagnation point of the flow field. Example 11 demonstrates hydrodynamic particle trapping.  FIGS. 18A-18C  shows a time series of three fluorescence micrographs of trapping and concentration control of fluorescent nanoparticles  1801  at different trapping conditions defined by the inflow rate of the flow-recirculating microfluidic device Immobile particles ( 1802 ) are unaffected by the flow. An exemplary use of fields in the invention is depicted in  FIG. 21A , where two membrane-attached objects  2102  and  2103  are brought together in order to bind them to each other  2105 . 
         [0138]    In one aspect, the control of the concentration is used to concentrate membrane-bound biomolecules, such as nuclear-receptors, or G-protein coupled receptors. In a related embodiment, the invention is used to concentrate membrane proteins in a membrane area so that they assume an ordered structure or crystallize.  FIG. 21E  shows an example of the arrangement of membrane proteins into an ordered two-dimensional assembly. 
         [0139]    The invention can be applied where positioning of a membrane-bound objects close to a sensor or probe is desired. Exemplary embodiments of such sensors or probes are chemical or optical sensors, or electrodes. In one aspect, trapping is used to exert a force onto a membrane-bound object, to measure forces acting on membrane-bound objects, to measure interactions between membrane-bound objects, or to measure interactions between membrane-bound objects and non-membrane-bound objects. An exemplary embodiment is the monitoring of allosteric interactions between membrane proteins ( FIG. 21A ). In another aspect, trapping is used to study mechanical properties of the membranes or membrane-bound objects. Exemplary embodiments are the measurement of DNA stretching, membrane rupturing, or adhesion strength. 
         [0140]    The invention can be used in combination with detection methods and devices. Such detectors can be utilized to analyze chemical or physical modifications or structural changes of the membrane, or material released from the membrane. In one embodiment, the system is coupled with an analytical detection mechanism. In one embodiment, the detection mechanism is electrical. In one embodiment, the detection is optical. In one embodiment, the detection mechanism is electrochemical. In one embodiment, the detection mechanism is mechanical. In one embodiment, the optical detection mechanism is microscopy-based. The invention can be applied in environments including an optical microscope. Optical microscopes can be both upright and inverted light microscopes. Examples of optical microscopes include fluorescence, epi-fluorescence, confocal, or TIRF microscopes. 
         [0141]    In one aspect, the invention includes temperature control in the vicinity of the membrane. In one aspect, the temperature control is global, wherein the temperature of the entire membrane is changed. In another aspect, the temperature control is locally applied to a selected membrane region. In one embodiment, temperature control is used to change the fluidity of the deposited membrane. In one aspect, the combination of temperature control and the phase transition of surfactants in the membrane is used for on/off-switching of transport and diffusion of membrane-attached objects. An exemplary embodiment uses thermotropic lipids as membrane source. 
         [0142]    In one embodiment, temperature control is used to change the composition of the membrane. In one aspect, the composition change of the membrane comprises phase transition and phase separation. In another aspect, the composition change of the membrane comprises lipid raft formation. In one embodiment, temperature control is used to modify the chemical reactivity of membrane components. In one embodiment, temperature control is used to modify the chemical reactivity of processing solutions. In one embodiment, temperature control is used to cause morphological changes of the membrane, or disintegrate the membrane. 
         [0143]    In one embodiment, temperature control is achieved by means of heating. Examples of heat sources are resistive heaters, Peltier elements, radiative heaters, continuous wave or pulsed laser heaters, and convective heaters. In one preferred embodiment, the temperature control is achieved with a resistive heater under the membrane ( FIG. 22F ), such that heat conduction  2214  through the substrate changes the temperature of the membrane  2202 . 
         [0144]    In one embodiment, the temperature control is used to establish a temperature gradient over the membrane or over a local area of the membrane. In one embodiment, the thermal gradient is used to transport membrane material by means of thermomigration or thermo diffusion. In one embodiment, temperature control is used in the vicinity of a stagnation point created by a field ( FIGS. 22A-22F ).  FIG. 22A  depicts an embodiment in which region of higher temperature ( 2202 ) is created in the vicinity of the stagnation point ( 2203 ), while a lower temperature region ( 2201 ) is maintained further away from it. The mobility factor is influenced by the temperature ( FIG. 22B ). This can be due to variable viscosity of the membrane, phase transition of the membrane, a transformation of the object or change in the interaction between the membrane and the object. In another aspect, the stagnation point and the point of local heating are positioned in the same location ( FIGS. 22A and 22B ). Local heating can be co-localized with the stagnation point. In one embodiment, radiation can be guided through an optical waveguide to affect the membrane locally ( 2209 ) ( FIG. 22C ). 
         [0145]    In some preferred embodiments, the waveguide can be attached to the field source. In some embodiments, the waveguide is an optical fiber. In some other embodiments, the waveguide is microfabricated into the flow-recirculating fluidic device ( 2203 / 2209 ). In another embodiment, the radiation can be provided through a microscope objective  2211  ( FIG. 22D ). In another embodiment, the temperature control is achieved with fluid circulation ( FIG. 22E ). An injection channel  2212  close to the aspiration channel  2203  is used to inject a fluid of higher temperature  2213  into the flow field around the aspiration channel  2204 , such that it reaches the membrane  2202 . 
       EXAMPLES 
       [0146]    Non-limiting examples of the invention are presented herein. 
       Experimental Setup 
     Microfluidic Device 
       [0147]    Aspiration control was achieved my means of a microfluidic device described in Ainla, et al. A multifunctional pipette, Lab Chip 2012 coupled to a pressure controller. The microfluidic device has the following properties and dimensions. Channel size: 30 μm×30 μm, channel-channel separation at the tip: 20 μm, channel-bottom separation at the tip: 20 μm, solution reservoirs: 35 μL, flow conductance of supply channels: 53 nL/(s*bar), outflow: 3.2 nL/s, inflow (from 2 channels): 10.6 nL/s, ratio (outflow/inflow): 0.3. The device has the capability to switch in a valveless fashion between four different solutions. 
         [0148]      FIGS. 11A and 11B  provide schematic views of this device ( 1101 ) and the utilization for the fabrication of a membrane ( 1104 ) on a surface ( 1102 ). The device comprises inflow ( 1106 ) and outflow ( 1105 ) channels, which generate a flow recirculation volume ( 1103 ). The inflow is selected through a valveless switching chamber ( 1107 ), comprising a number of supply ( 1108 ) and vacuum ( 1110 ) channels.  FIG. 11A  is a schematic top down view.  FIG. 11B  a perspective view. 
       Micropositioning 
       [0149]    Micropositioning was implemented using manual water hydraulic micromanipulators (Narishige MH-5, Japan) or electronic computer controllable micromanipulators (Scientifica PatchStar, UK). The micromanipulators allow positioning of the pipette and bringing the tip into proximity of the desired objects of interest inside the reservoir. 
         [0150]    The experimental setup comprised the multifunctional pipette, a laser scanning confocal microscope Leica IRE2 (Leica Microsystems GmbH, Wetzlar, Germany) equipped with Leica TCS SP2 confocal scanner with AOBS™ and Ar/ArKr and HeNe lasers to provide excitation wavelength 488, 594 and 633 nm. Objectives used were HC PL APO CS 20×0.70 UV and HCX PL APO CS 40×1.25 OIL UV. The sample position was controlled by a scanning stage SCAN IM 120×100 (Märzhäuser Wetzlar GmbH &amp; Co. KG, Wetzlar, Germany), equipped with a CORVUS™ stage controller (Märzhäuser). Both scanning stage and pipette control unit were connected to a PC computer via USB port. Custom software, written in Microsoft Visual C++ (.NET), allowed simultaneous control of stage position and pipette control unit, through which the liquid composition and deposition spot size were controlled. The pipette was held and positioned in the beginning of an experiment by a 3-axis water hydraulic micromanipulator Narishige MH-5 (Japan). During the experiment, the pipette tip was positioned about ˜10-20 μm above the surface, so that materials could be delivered to the surface, while avoiding direct contact, which would damage the lipid film. 
       Experiments 
     Surfaces 
     Glass Surfaces 
       [0151]    Circular microscope cover glasses #1.5 (Menzel-Gläser, 47 mm diameter) were obtained from Thermo Scientific (Sweden). Before use, the glass surfaces were cleaned in the MC2 process laboratory at Chalmers University of Technology. First, the slides were immersed in freshly prepared piranha solution (3:1 v/v mixture of concentrated H 2 SO 4  and 30% H 2 O 2 , heated to 100-110° C.) for 10 min, followed by rinsing with deionixed water and blow drying with nitrogen. Thereafter, the glass slices were mounted to a WILLCO WELLS™ dish frame using a dedicated double sided tape and assembly kit (Willco Wells B. V., Amsterdam, Netherlands) and stored in a sealed plastic bag until use. 
       SU-8 Surfaces 
       [0152]    The cleaned cover glasses were coated with ˜2 μm high SU-8 patterns using the procedure provided by Microchem Corporation. SU-8 2002 (Microchem Corp, Massachusetts, USA) was spin-coated at 3000 rpm for 30 s, followed by soft baking for 2 min at 95° C. on a hot-plate. The SU-8 film was exposed with a dose of 120 mJ/cm 2  on a Karl-Süss™ contact mask aligner MA6 (G-line, 5-6 mW/cm 2 ), using the “Low-Vac” mode with a bright-field chromium mask. The substrates were then post-exposure-baked for 2 min at 95° C. on a hot-plate. Thereafter, the SU-8 was developed in SU-8 Developer (Microchem) for 1 min using two sequential bathes, rinsed by spraying with clean developer, and blow dried with nitrogen, yielding a SU-8 coated cover glass where the channels are formed by the exposed glass. The surfaces were plasma cleaned briefly in a Plasma Therm BatchTop RIE (50 W, 250 mTorr, 1 min) plasma chamber, and hard baked for 10 min at 200° C. on a hot plate with slow heating and cooling to prevent crack formation. The so-prepared glass slides were mounted to dish frames like the plain glass slides described in the previous section. 
       Lipid Source Preparation 
       [0153]    The following vesicle compositions were used: 
         [0154]    POPC-488: POPC 99%, ATTO88-DOPE 1%; 
         [0155]    POPC-655: POPC 99%, ATTO655-DOPE 1%; 
         [0156]    POPC-B: POPC 99%, Biotin-PE 1%; 
         [0157]    POPC-488B: POPC 98%, ATTO488-DOPE 1%, Biotin-PE 1%; 
         [0158]    POPC-655B: POPC 98%, ATTO655-DOPE 1%, Biotin-PE 1%; and 
         [0159]    DOTAP-655: PC 49%, DOTAP 50%, ATTO655-DOPE 1%. 
         [0160]    1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Soy L-α-phosphatidylcholine (PC), 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotin-PE) were obtained from Avanti Polar Lipids (USA). ATTO 488 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (ATTO 488-DOPE) and ATTO 655 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (ATTO 655-DOPE) were provided by ATTO-TEC GmbH (Germany). 
       Stock Vesicle Suspensions 
       [0161]    For each recipe, a designated amount (see above) of lipids and lipid conjugates in chloroform were mixed and diluted with chloroform to a total concentration of 10 mg/ml. 300 μl of this solution was placed in a 10 ml round bottom flask, and the chloroform was removed in a rotary evaporator at reduced pressure (−80 kPa) over a period of 6 hours. The dry lipid film at the walls of the flask was rehydrated with 3 ml of PBS buffer containing 5 mM Trisma Base (Sigma Aldrich), 30 mM K 3 PO 4  (Sigma Aldrich), 30 mM KH 2 PO 4  (Sigma Aldrich), 3 mM MgSO 4 *7H 2 O (Merck), and 0.5 mM Na 2 EDTA (Sigma Aldrich). The pH was adjusted to 7.4 with H 3 PO 4  (Sigma Aldrich). The rehydrated lipid cake was placed in the fridge (4° C.) overnight. In the final step, the lipid cake was sonicated at 120 W/35 kHz (Bandelin Sonorex, Germany) at room temperature for 15-30 s, to induce the formation of giant vesicles of varying, mainly multiple lamellarity. 
       Small Unilamellar Vesicles 
       [0162]    Small unilamellar vesicles were prepared on the day the experiments were conducted. 100 μl of the desired vesicle stock solutions were diluted (1:10) with TRIS buffer [125 mM NaCl (Sigma Aldrich), 10 mM TRIS (VWR), 1 mM Na 2 EDTA (Sigma Aldrich), adjusted to pH=7.4 and sonicated using a Sonics &amp; Materials Vibra Cell™ High Intensity Ultrasonic Liquid Processor (Model 501, CIAB, Chemical Instruments AB, Sweden)] at 15° C. for 10 minutes. The sonicated samples were subsequently ultra-centrifuged at 40,000 rpm at 15° C. for 30 minutes to separate multilamellar aggregates and tip debris (Beckman TL-100 Ultracentrifuge, USA). The small unilamellar vesicles in the supernatant were transferred to a separate tube. 
       Triton X 
       [0163]    Triton X detergent was obtained from Sigma Aldrich and diluted 1:10 with TRIS buffer. 
       Antibodies 
       [0164]    Antibodies were obtained from Agrisera (Sweden). 0.2 mg of goat anti-biotin antibody conjugated to DyLight 650 (“Goat anti-biotin”) was dissolved in 1 ml of 10 mM TRIS buffer. 0.2 mg of donkey anti-Goat IgG antibody conjugated to DyLight 594 (“Anti-goat”) was dissolved in 1 ml of 10 mM TRIS buffer. All antibody solutions were prepared instantly prior to the experiments, at room temperature. 
       Biotin Solution 
       [0165]    1 mg powder of ATTO 488-biotin (Sigma Aldrich, Mo., USA) was diluted with 5 ml of HEPES buffer (10 mM HEPES, 100 mM NaCl, pH=7.4 adjusted with NaOH) to a final concentration of 0.2 mg biotin/ml HEPES. 
       Particle Solution 
       [0166]    8 μl of NEUTRAVIDIN®-coated latex particles (NEUTRAVIDIN® Labeled Microspheres, 0.2 μm, Yellow-Green Fluorescent (505/515), 1% solids, Invitrogen (Life Technologies); CA, USA) was diluted with 392 μl of HEPES buffer (10 mM HEPES, 100 mM NaCl, pH=7.4). The diluted solution is sonicated for 15 minutes (sonication frequency: 35 kHz, sonication power: 30/120 W, Bandelin Sonorex, Germany) and filtered through a PVDF (Hydrophilic polyvinylidene fluoride) membrane (Acrodisc LC syringe filter with effective filtration area of 13 mm with 0.2 μm pore size, PALL Life Sciences; NY, USA). 
       Example 1 
     Deposition of a POPC Spot (FIG.  12  A-C) 
       [0167]    The flow-recirculating microfluidic device was loaded with POPC-488 as membrane source. The device was positioned and the flow of lipid vesicle suspension (POPC-488) was switched-on for 160 s ( FIG. 12  A). A uniformly fluid lipid membrane was formed with a time constant of ˜20 s. The covered area remained nearly constant ( FIG. 12  B).  FIG. 12C  shows the exponential surface coverage kinetics with a time constant of about 20 s. The inset demonstrates spot uniformity. When the surface becomes covered, it eventually forms a uniform film. Density and size evolution of the spot were analysed. 
       Example 2 
     Deposition of a DOTAP Spot (FIGS.  12 D-F) 
       [0168]    The flow-recirculating microfluidic device was loaded with DOTAP-655 as membrane source. The device was positioned and the flow of lipid vesicle suspension (DOTAP-655) was switched-on for 600 s ( FIGS. 12D-E ). A uniformly fluid lipid membrane was formed immediately. The covered area grows continuously with a linear increase in spot radius. ( FIG. 12F ). Density and size evolution of the spot were analyzed. 
       Example 3 
     Multiplexing of Different Membrane Sources (FIGS.  12 G-I) 
       [0169]    Switching between different membrane sources was used to create membranes with a variable composition. The flow-recirculating microfluidic device was loaded with two different membrane sources (POPC-488, POPC-655). The pipette was positioned and the flow of lipid vesicle suspension was started. Pulse width modulation was used to multiplex the flow according to the desired composition. The composition was changed over time in steps of 10%.  FIGS. 12G-H  show the fluorescence emission channels of each membrane component.  FIG. 12I  (normalized emission intensity vs. PWM ratio) quantifies this development. 
       Example 4 
     Membrane Fusion (FIG.  12 J) 
       [0170]    The flow-recirculating microfluidic device was loaded with two types of lipid vesicles (POPC-488 and DOTAP-655). Thereafter, the pipette was positioned and the flow of POPC-488 vesicle suspension was switched on for 60 s to deposit a POPC spot. Thereafter, the flow was switched off and the pressure conditions were set such that the outflow rate would be about half the previous rate, to ensure that the size of the hydrodynamically confined flow (HCF) volume is reduced and the DOTAP is deposited within the boundaries of the already existing POPC film. Thereafter, DOTAP-655 deposition was switched on. The DOTAP membrane source fused into the previously formed membrane, resulting in an increase in the patch size due to spreading. 
       Example 5 
     Surface Writing (FIG.  13 ) 
       [0171]    The flow-recirculating microfluidic device was loaded with POPC-655 as a membrane source. The device was positioned and the flow of membrane source (POPC-655) was switched-on. Three written membrane geometries (heart, stickman, and smile, as shown in  FIGS. 13A-C , respectively) were fabricated by computer controlled translation of the surface. Step size of the motion was 13-18 μm and membrane was deposited in each step for 7 s. Since the spot diameter was about 100 μm, the line area was deposited in about 30 s. 
       Example 6 
     Multicomponent Surface Writing 
       [0172]    Multicomponent surface writing of two partially overlapping membrane patches is depicted in  FIGS. 13D-E . The flow-recirculating microfluidic device was loaded with two different membrane sources (POPC-488, POPC-655). The device was positioned and the flow of lipid vesicle suspension was started. Two 300 μm long and 100 μm wide lanes were written with a step size of 25 μm and a deposition time of 10 s. First, POPC-488 was written, followed by POPC-655. The lanes were offset by 50 μm and were overlapping partially ( FIG. 13  D). After writing, the diffusion was monitored for 15 min and the fluorescence intensity vs. a linear coordinate through the written areas (arrow) was analyzed ( FIG. 13E ). 
       Example 7 
     Membrane Removal (FIG.  14 ) 
       [0173]    The flow-recirculating microfluidic device was loaded with three solutions: two types of membrane source (POPC-488 and POPC-655) and a solution of dilute Triton-X. Two parallel lanes of 150 μm length were written ( FIG. 14A ), using POPC-655. Thereafter Triton-X solution was switched on (using increased supply pressure to compensate higher viscosity). The HCF volume is easily visible in the transmission channel of the microscope, due to a higher refractive index ( FIG. 14B ). Triton-X was scanned perpendicularly over the lower lane ( FIG. 14C ). Thereafter, the outflow was switched off again, the supply pressure was set to its initial value, and the pipette was positioned onto the cutting point of the lane. A spot of POPC-488 was deposited in order to reconnect the lane ( FIG. 14  D-E). 
       Example 8 
     Sequential Membrane Functionalization (FIGS.  15 A- 15 D) 
       [0174]    The flow-recirculating microfluidic device was loaded with four solutions: two types of membrane source (POPC-488B and POPC-B) and two antibody (primary goat anti biotin and secondary anti goat) solutions. The device was positioned and a 200 μm lane was written with a step size of 10 μm and a deposition time of 5 s, such that first 100 μm were composed of POPC-488B, followed by 100 μm of POPC-B ( FIG. 15A ). Thereafter, the device was moved 100 μm backwards onto the middle point of the lane, and goat anti-biotin was applied for 5 s, followed by 10 min diffusion time ( FIG. 15B ). Thereafter anti-goat was applied for 5 s onto the same spot ( FIG. 15C ), after which re-distribution of molecular species was monitored for about 15 min ( FIG. 15D ). 
       Example 9 
     Hydrodynamic Vesicle Trapping 
       [0175]    The flow-recirculating microfluidic device was loaded with two different membrane sources (POPC-488, DOTAP-655). This experiment was started by depositing POPC membrane source onto a DOTAP membrane ( FIG. 16A ). After the POPC membrane source was deposited onto the DOTAP film, the supply pressures were adjusted for maximal inflow (Q≈33 nl/s) through the middle channel ( FIG. 16B ). Trap formation and collection of vesicles under the middle channel were monitored ( FIGS. 16C and 16D ). 
       Example 10 
     Controlled Flow in Patterned 2D Channel 
       [0176]    Flow-recirculating microfluidic device was loaded with two different membrane sources (POPC-488, DOTAP-655) and positioned onto the SU-8 patterned area ( FIG. 17A ). First, the surface of the patterned channel was covered with POPC-488, thereafter DOTAP-655 was deposited onto the circular supply area, and transport of fluorescently labelled lipid from the supply area into the channel was monitored for about 25 min ( FIG. 17B-C ). 
       Example 11 
     Hydrodynamic Particle Trapping 
       [0177]    The surface was approached with the flow-recirculating microfluidic device. NEUTRAVIDIN®-coated particles were injected through the outflow channel of the device, followed by a resting time of ˜10 seconds. Subsequently, biotin solution was injected through the same channel after internal switching between the two solutions into the vicinity of the lipid membrane patch. This biotin blocking of the remaining free binding sites of avidin prevents the encapsulation of the latex particles by the biotinylated lipid membrane. After adjustment of the flow parameters of the pipette, accumulation of particles in a confined area around the stagnation point is observed on the lipid patch. The particles migrate on the surface of the membrane, following the movement of the stagnation point created by the pipette.  FIG. 18  shows a series of fluorescence micrographs of this trapping and concentration control experiment. 
       Example 12 
     Deposition of a Lipid Monolayer (FIG.  23 ) 
       [0178]    Conductive indium tin oxide (ITO) coated cover glass slides were coated with 80 nm of DUPONT® TEFLON® AF amorphous fluoropolymer by spin-coating the coated surfaces and were e-beam patterned, using a JEOL JBX9300 e-beam lithography system, an acceleration voltage of 100 kV, and an exposure dose of 1000 μC/cm 2 . The exposed pattern defined the edge of the area on which a lipid monolayer is able to spread. The patterned cover glass-slides were submerged to aqueous TRIS buffer under a confocal microscope (as described in the previous example). The flow-recirculating microfluidic device was loaded with DOTAP-655 as membrane source. The device was positioned on the top of deposition area encompassed by the exposed spreading barrier. The flow of lipid vesicle suspension was switched on for 20 min ( FIG. 23 ). A continuous fluid lipid monolayer was formed, covering the entire available unexposed deposition area. 
       INCORPORATION BY REFERENCE 
       [0179]    The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated by herein in their entireties by reference. 
       EQUIVALENTS 
       [0180]    The functions of several elements may, in alternative embodiments, be carried out by fewer elements, or a single element. Similarly, in some embodiments, any functional embodiment may perform fewer, or different, operations than those described with respect to the illustrated embodiments. Also, functional elements shown as distinct for purposes of illustration may be incorporated within other functional elements separated in different hardware or distributed in a particular implementation. 
         [0181]    While certain embodiments according to the invention have been described, the invention is not limited to just the described embodiments. Various changes and/or modifications can be made to any of the described embodiments without departing from the spirit or scope of the invention. Also, various combinations of elements, steps, features, and/or aspects of the described embodiments are possible and contemplated even if such combinations are not expressly identified herein.