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  Systems and methods for rapidly changing the solution environment around sensorsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Viable Micro-organismThe Patent Description & Claims data below is from USPTO Patent Application 20060078961. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The invention relates to systems and methods for rapid and programmable delivery of aqueous streams to a sensor, such as a cell-based biosensor. In particular, the invention provides methods and systems for high throughput patch clamp analysis. BACKGROUND OF THE INVENTION [0002] Ion-channels are important therapeutic targets. Neuronal communication, heart function, and memory all critically rely upon the function of ligand-gated and voltage-gated ion-channels. In addition, a broad range of chronic and acute pathophysiological states in many organs such as the heart, gastrointestinal tract, and brain involve ion channels. Indeed, many existing drugs bind receptors directly or indirectly connected to ion-channels. For example, anti-psychotic drugs interact with receptors involved in dopaminergic, serotonergic, cholinergic and glutamatergic neurotransmission. [0003] Because of the importance of ion-channels as drug targets, there is a need for methods which enable high throughput screening (HTS) of compounds acting on ligand-gated and voltage-gated channels (see e.g., Sinclair et al., 2002, Anal. Chem. 74: 6133-6138). However, existing HTS drug discovery systems targeting ion channels generally miss significant drug activity because they employ indirect methods, such as raw binding assays or fluorescence-based readouts. Although as many as ten thousand drug leads can be identified from a screen of a million compounds, identification of false positives and false negatives can still result in a potential highly therapeutic blockbuster drug being ignored, and in unnecessary and costly investments in false drug leads. [0004] Patch clamp methods are superior to any other technology for measuring ion channel activity in cells, and can measure currents across cell membranes in ranges as low as picoAmps (see, e.g., Neher and Sakmann, 1976, Nature 260: 799-802; Hamill, et al., 1981, Pflugers Arch 391: 85-100; Sakmann and Neher, 1983, In Single-Channel Recording pp. 37-52, Eds. B. Sakmann and E. Neher. New York and London, Plenum Press, 1983). However, patch clamp methods generally have not been the methods of choice for developing HTS platforms. SUMMARY OF THE INVENTION [0005] The invention provides microfluidic systems for altering the solution environment around a nanoscopic or microscopic object, such as a sensor, and methods for using the same. The invention can be applied in any sensor technology in which the sensing element needs to be exposed rapidly, sequentially, and controllably, to a large number of different solution environments (e.g., greater than 10 and preferably, greater than about 96 different environments) whose characteristics may be known or unknown. In contrast to prior art microfluidic systems, the interval between sample deliveries is minimized, e.g., on the order of microseconds and seconds, permitting rapid analysis of compounds (e.g., drugs). [0006] In one aspect, the invention provides a system comprising a substrate for changing the solution environment around a nanoscopic or microscopic object, such as a sensor. The substrate comprises an open-volume chamber for the sensor, and a plurality of channels. Each channel comprises an outlet for delivering a substantially separate aqueous stream into the chamber. In one aspect, the outlets are substantially parallel, i.e., arrayed linearly in a single plane. The dimensions of the outlets can vary; however, in one aspect, where the sensor is a biological cell, the diameter of each of the outlets is, preferably, at least about the diameter of the cell. Preferably, a plurality, if not all, of the channels programmably deliver a fluid stream into the chamber. [0007] In a preferred aspect, each channel of the substrate comprises at least one inlet for receiving solution from a reservoir, conforming in geometry and placement on the substrate to the geometry and placement of wells in a multi-well plate. For example, the substrate can comprise 96-1024 reservoirs, each connected to an independent channel on the substrate. Preferably, the center-to-center distance of each reservoir corresponds to the center-to-center distance of wells in an industry standard microtiter or multi-well plate. [0008] In a further aspect, the substrate comprises one or more treatment chambers or microchambers for delivering a treatment to a cell placed within the treatment chamber. The treatment can comprise exposing the cell to a chemical or compound, (e.g. drugs or dyes, such as calcium ion chelating fluorogenic dyes), exposing the cell to an electrical current (e.g., electroporation, electrofusion, and the like), or exposing the cell to light (e.g., exposure to a particular wavelength of light). A treatment chamber can be used for multiple types of treatments which may be delivered sequentially or simultaneously. For example, an electrically treated cell also can be exposed to a chemical or compound and/or exposed to light. Treatment can be continuous over a period of time or intermittent (e.g., spaced over regular or irregular intervals). The cell treatment chamber can comprise a channel with an outlet for delivering a treated cell to the sensor chamber or directly to a mechanism for holding the cell connected to a positioner (e.g., a micropositioner or nanopositioner) for positioning the cell within the chamber. [0009] Preferably, the base of the sensor chamber is optically transmissive and in one aspect, the system further comprises a light source (e.g., such as a laser) in optical communication with the open volume chamber. The light source can be used to continuously or intermittently expose the sensor to light of the same or different wavelengths. The sensor chamber and/or channels additionally can be equipped with control devices. For example, the sensor chamber and/or channels can comprise temperature sensors, pH sensors, and the like, for providing signals relating to chamber and/or channel conditions to a system processor. [0010] The sensor chamber can be adapted for receiving a variety of different sensors. In one aspect, the sensor comprises a cell or a portion of a cell (e.g., a cell membrane fraction). In another aspect, the cell or cell membrane fraction comprises an ion channel, including, but not limited to, a presynaptically-expressed ion channel, a ligand-gated channel, a voltage-gated channel, and the like. In a further aspect, the cell comprises a receptor, such as a G-Protein-Coupled Receptor (GPCR), or an orphan receptor for which no ligand is known, or a receptor comprising a known ligand. [0011] A cultured cell can be used as a sensor and can be selected from the group consisting of CHO cells, NIH-3T3 cells, and HEK-293 cells, and can be recombinantly engineered to express a sensing molecule such as an ion channel or receptor. Many other different cell types also can be used, which can be selected from the group consisting of mammalian cells (e.g., including, but not limited to human cells, primate cells, bovine cells, swine cells, other domestic animals, and the like); bacterial cells; protist cells; yeast cells; plant cells; invertebrate cells, including insect cells; amphibian cells; avian cells; fish; and the like. [0012] A cell membrane fraction can be isolated from any of the cells described above, or can be generated by aggregating a liposome or other lipid-based particle with a sensing molecule, such as an ion channel or receptor, using methods routine in the art. [0013] The cell or portion of the cell can be positioned in the chamber using a mechanism for holding the cell or cell portion, such as a pipette (e.g., a patch clamp pipette) or a capillary connected to a positioner (e.g., such as a micropositioner or nanopositioner or micromanipulator), or an optical tweezer. Preferably, the positioner moves the pipette at least in an x-, y-, z-, direction. Alternatively or additionally, the positioner may rotate the pipette. Also, preferably, the positioner is coupled to a drive unit which communicates with a processor, allowing movement of the pipette to be controlled by the processor. [0014] In one aspect, the base of the chamber comprises one or more depressions and the cell or portion of the cell is placed in a depression which can be in communication with one or more electrodes (e.g., the sensor can comprise a planar patch clamp chip). [0015] Non-cell-based sensors also can be used in the system. Suitable non-cell based sensors include, but are not limited to: a surface plasmon energy sensor; an FET sensor; an ISFET; an electrochemical sensor; an optical sensor; an acoustic wave sensor; a sensor comprising a sensing element associated with a Quantum Dot particle; a polymer-based sensor; a single molecule or an array of molecules (e.g., nucleic acids, peptides, polypeptides, small molecules, and the like) immobilized on a substrate. The sensor chamber also can comprise a plurality of different types of sensors, non-cell based and/or cell-based. A sensor substrate can be affixed to the base of the chamber or the substrate can simply be placed on the base of the chamber. Alternatively, the base of the chamber itself also can serve as the sensor substrate and one or more sensing elements can be stably associated with the base using methods routine in the art. In one aspect, sensing elements are associated at known locations on a substrate or on the base of the sensor chamber. [0016] However, an object placed within a chamber need not be a sensor. For example, the object can be a colloidal particle, beads, nanotube, a non-sensing molecule, silicon wafer, or other small elements. [0017] The invention also provides a system comprising a substrate which comprises at least one chamber for receiving a cell-based biosensor, a plurality of channels, at least one cell storage chamber and at least one cell treatment chamber. Preferably, each channel comprises an outlet for delivering a fluid stream into the chamber, and the cell treatment chamber is adapted for delivering an electrical current to a cell placed within the cell treatment chamber. In one aspect, the cell treatment chamber further comprises a channel with an outlet for delivering a cell to the sensor chamber for receiving the cell-based biosensor. The system can be used to rapidly and programmably change the solution environment around a cell which has been electroporated and/or electrofused, and/or otherwise treated within the cell treatment chamber. Alternatively, or additionally, the sensor chamber also can be used as a treatment chamber and in one aspect, the sensor chamber is in electrical communication with one or more electrodes for continuously or intermittently exposing a sensor to an electric field. [0018] In one aspect, a system according to the invention further comprises a scanning mechanism for scanning the position of a sensor relative to the outlets of the channels. The scanning mechanism can translate the substrate relative to a stationary sensor, or can translate the sensor relative to a stationary substrate, or can move both sensor and substrate at varying rates and directions relative to each other. In one aspect, the sensor is positioned relative to an outlet using a mechanism for holding the sensor (e.g., such as a pipette or capillary) coupled to a positioner (e.g., a micropositioner or nanopositioner or micromanipulator). Thus, the positioner can be used to move the sensor across a plurality of fluid streams exiting the outlets of the channels by moving the mechanism for holding the sensor. Alternatively, or additionally, scanning also can be regulated by producing pressure drops sequentially across adjacent microchannels. [0019] Preferably, the scanning mechanism is in communication with a processor and translation occurs in response to instructions from the processor (e.g., programmed instructions or instructions generated as a result of a feedback signal). In one aspect, the processor controls one or more of: the rate of scanning, the direction of scanning, acceleration of scanning, and number of scans. Thus, the system can be used to move nanoscopic and microscopic objects in a chamber to user-selected, or system-selected coordinates, for specified (e.g., programmable) lengths of time. Preferably, the system processor also can be used to locate the position of one or more objects in the chamber, e.g., in response to previous scanning actions and/or in response to optical signals from the objects detected by the system detector. In one aspect, the system further comprises a user device in communication with the processor which comprises a graphical user display for interfacing with a user. For example, the display can be used to display coordinates of object(s) within the chamber, or optical data or other data obtained from the chamber. [0020] The invention additionally provides a substrate comprising a chamber for receiving a cell-based biosensor which comprises a receptor or ion channel. In one aspect, the system sequentially exposes a cell-based biosensor for short periods of time to one or several ligands which binds to the receptor/ion channel and to buffer without ligand for short periods of time through interdigitated channels of the substrate. For example, selective exposure of a cell biosensor to these different solution conditions for short periods of time can be achieved by scanning the cell-based biosensor across interdigitated channels which alternate delivery of one or several ligands and buffer. The flow of buffer and sample solution in each microfluidic channel is preferably a steady state flow at constant velocity. However, in another aspect, the system delivers pulses (e.g., pulsatile on/off flow) of buffer to a receptor through a superfusion capillary positioned in proximity to both the cell-based biosensor or other type of sensor and to an outlet through which a fluid is streaming. For example, the system can comprise a mechanism for holding the sensor which is coupled to a positioner (e.g., a micropositioner, nanopositioner, micromanipulator, etc.) for positioning the c sensor in proximity to the outlet and a capillary comprising an outlet in sufficient proximity to the mechanism for holding the sensor to deliver a buffer from the capillary to the sensor. A scanning mechanism can be used to move both the capillary and sensor simultaneously, to maintain the appropriate proximity of the capillary to the sensor. The capillary also can be coupled to a pumping mechanism to provide pulsatile delivery of buffer to the sensor. In another aspect, the flow rate of buffer from the one or more superfusion capillaries in proximity to one or more sensors can be higher or lower than the flow rate of fluid from the channels. [0021] The invention further provides a substrate which comprises a circular chamber for receiving a sensor, comprising a cylindrical wall and a base. In one aspect, the substrate comprises a plurality of channels comprising outlets whose openings are radially disposed about the circumference of the wall of the chamber (e.g., in a spokes-wheel configuration), for delivering samples into the chamber. Preferably, the substrate also comprises at least one output channel for draining waste from the chamber. In one aspect, at least one additional channel delivers buffer to the chamber. Preferably, the angle between the at least one additional channel for delivering buffer and the output channel is greater than 100. More preferably, the angle is greater than 90.degree.. The channel "spokes" may all lie in the same plane, or at least two of the spokes may lie in different planes. Continue reading... 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