Systems and methods for rapidly changing the solution environment around sensors

Abstract

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 whose characteristics may be known or unknown.

Description

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 whic...

AI Technical Summary

Benefits of technology

[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).

[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.

[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.

[0027] The system can thus regulate when, and through which channel, a fluid stream is withdrawn from the chamber. For example, after a defined period of time, a fluid stream can be withdrawn from the chamber through the same channel through which it entered the system or through a different channel. When a drain channel is adjacent to a delivery channel, the system can generate a U-shaped fluid stream which can efficiently recycle compounds delivered through delivery channels.

[0039] In another aspect, the viscosity of fluids in at least two of the channels is different. In yet another aspect, fluid within at least two of the channels are at a different temperature. In a further aspect, the osmolarity of fluid within at least two of the channels is different. In a still further aspect, the ionic strength of fluid within at least two of the channels is different. Fluid in at least one of the channels also can comprise an organic solvent. By changing these parameters at different outlets, sensor responses can be optimized to maximize sensitivity of detection and minimize background. In some aspects, parameters also can be varied to optimize certain cell treatments being provided (e.g., such as electroporation or electrofusion).

[0048] In one aspect, a periodically resensitized receptor is provided using the superfusion system described above to deliver pulses of buffer to the cell-based biosensor, to thereby remove any bound agonist or modulator desensitizing the receptor, before the receptor is exposed to the next channel outlet containing agonists or receptor modulators. In detection of antagonists, the pulsated superfusion system can also periodically remove the constantly applied agonist. A transient peak response (which is desensitized to a steady state response) is generated when the resensitized biosensor is exposed to the agonist. The generation of this peak response can provide a better signal-to-noise ratio in detection of antagonists.

Problems solved by technology

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.

However, patch clamp methods generally have not been the methods of choice for developing HTS platforms.

Images