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Methods and apparatus for the manipulation of particle suspensions and testing thereof

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20120264134 patent thumbnailZoom

Methods and apparatus for the manipulation of particle suspensions and testing thereof


Apparatus and methods are provided for analysis of individual particles in a microfluidic device. The methods involve the immobilization of an array of particles in suspension and the application of experimental compounds. Such methods can also include electrophysiology studies including patch clamp recording, electroporation, or both in the same microfluidic device. The apparatus provided includes a microfluidic device coupled to a multi-well structure and an interface for controlling the flow of media within the microchannel device.
Related Terms: Electrophysiology

Browse recent Fluxion Biosciences Inc. patents - San Francisco, CA, US
Inventors: Cristian Ionescu-Zanetti, Michelle Khine, Michael Schwartz
USPTO Applicaton #: #20120264134 - Class: 435 613 (USPTO) - 10/18/12 - Class 435 


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The Patent Description & Claims data below is from USPTO Patent Application 20120264134, Methods and apparatus for the manipulation of particle suspensions and testing thereof.

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CROSS-REFERENCE

This application is a continuation of pending U.S. patent application Ser. No. 11/690,831, filed Mar. 25, 2007, which application claims the benefit of U.S. Provisional Application No. 60/744,034, filed Mar. 31, 2006, U.S. Provisional Application No. 60/868,864 filed Dec. 6, 2006, and U.S. Provisional Application No. 60/870,842 filed Dec. 19, 2006; these applications are incorporated herein by reference, in their entirety, for any purpose.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funds used to support some of the studies disclosed herein were provided by grant number 1 R43 GM075509-01 awarded by the National Institutes of Health from the National Institute for General Medical Sciences. The Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

The fields of flow cytometry, ion-channel electrophysiology, single cell electroporation, controlled shear force in vivo-simulating cell culture and numerous related biotechnology approaches stand to benefit from advances in the design of microfluidic devices for manipulation of cells and attendant apparatus.

Flow cytometry is a widely used technique for the counting and classification of single cells (Cottingham 2005). Because of its high throughput (1000 s of cells/s) and reliability, it has become the most widely used method for cell population identification. It has far reaching applications, from pharma and academic research to drug cell based screening and cell line QC. In the diagnostic market it is used primarily for the quantification of blood cell content, and used to monitor RBC counts as well as counts for all of the major WBC types (blood panel).

While flow cytometry was originally based on electrical resistance changes in a flow capillary, almost all modern flow cytometers are now based on a laser for excitation and PMT for the detection of fluorescent signals from each cell, thus providing data which is multiplexed with straight cell count data. This allows for the identification and counting of a number of different cell types (through the use of fluorescent probes), as well as establishing relations between the fluorescence intensities recorded.

A key drawback of this technique is the loss of sub cellular information. Cell morphological parameters, as well as localization of fluorescent signals w/in the cell and the correlation between various stains are all lost in a process which simply integrates the fluorescence intensity over the whole cell and outputs one number per cell.

Until recently, this type of information has only been accessible through the use of Image Scanning Cytometry, basically a microscope on an XYZ stage that scans a thin cavity filled w/cellular suspension. This is an automation of the manual hemocytometer. This technique is significantly slower as compared to flow cytometry, and requires automatic focus and due to movement of the substrate in the XY plane. An additional drawback of this technique is the inability to sort cells.

Recently introduced imaging flow cytometers aim to combine the speed and ease of use of flow cytometry with the high information content of Image Scanning Cytometry (Bonetta 2005). The instrument images cells as they flow by at high velocity in a single file. The requirement of assembling in-flight images of single cells requires a great deal of custom technical development, which translates in a relatively high price for such devices (approx. $300 k for ImageStream 100) and relegates their use to core labs and pharmaceutical companies.

Ion channels are functional units of all living systems and fulfill a number of roles, from fast signal transmission in the nervous system to regulation of biochemical pathways. In turn, a number of disorders have been linked to ion channel malfunction. Ion channels are implicated in mental disorders such as Alzheimer\'s and epilepsy, as well as heart disease, diabetes and neuromuscular disease [Shaffer]. Consequently, these transmembrane proteins are attractive drug targets and constitute about 20-30% of new drug development campaigns [Southhan].

The electrophysiology recording technique, termed patch clamp, has emerged as the gold standard in the study of ion channel function. It is based on the ability to perform recordings of transmembrane currents through a specific ion channel type. Traditionally, patch clamp recording is accomplished with a micromanipulator-positioned glass pipette under a microscope [Sackmann]. This technique was perfected in 1981-\'83 by Nether and Sackmann through the achievement of high resistance seals between the glass pipette tip and the cell membrane. The basic setup is illustrated in FIG. 5A. Current that passes through the ion channels in either the membrane patch or the whole cell membrane is recorded at different bias voltages.

In June 2005, the FDA mandated that all drugs must be tested against the potassium (K) ion channel hERG, whose unpredicted adverse modulation by several blockbuster drugs has been implicated in long-QT syndrome and subsequent sudden death by heart malfunction [Denyer]. It has been estimated that approximately 25-40% of all lead compounds show hERG activity in vitro [Bennett].

The gold standard assay for hERG safety screening is the patch clamp: the cell is voltage clamped in whole-cell configuration (using a glass pipette) while the test compounds are introduced extracellularly. The response of the cell to the test compounds is evident from the current response of the cell when the compounds have reached the ion channel\'s binding site which, in the case of hERG, is on the interior portion of the cell (see FIG. 6). FIG. 6 shows an outward rectifying potassium channel. As indicated in the diagram, the candidate hERG blocker (e.g., a compound to be tested) binds at the intracellular side of the molecule. Source: Enal Razvi [Southhan]. The problem with this approach is that the compounds are notoriously slow to diffuse through the membrane to reach this binding site (t>20 minutes). In addition, multiple compound concentrations need to be applied sequentially to the same patched cell in order to provide consistent measurement. Consequently, hERG measurements often fail due to the lack of long term stability for the high resistance seal between the glass pipette and the cell (seals often degrade in t<20 minutes).

Despite constant improvement of the traditional patch clamp technique, it remains laborious, requiring pipettes to be placed in the cell vicinity by a skillful operator using a micromanipulator under a microscope. Consequently, the patch clamp technique has been difficult to use in drug development, where high-throughput automated measurements are required. An automated patch clamp setup for high-throughput measurements using disposable devices would eliminate the prohibitive time investment of the traditional patch clamp, while maintaining its advantages over indirect measurements of ion channel behavior. The first approach to automated patch consisted of an array of robotically operated patch clamp pipettes (Axon, Inc.), to be used with large cells (Xenopus oocytes). The most serious drawbacks of this approach its inability to work with mammalian cell lines as well as the complexity of the manipulation system, while savings in terms of reagent use are minimal. A microfabricated patch clamp approach, if perfected, would solve both these problems. Currently available automated electrophysiology devices are employed by large organizations at large capital expense (greater than $400,000 per instrument) as well as a large cost per data point (about $10 per cell trap). They also retain important limitations in the area of optical observation and compound perfusion.

RNA interference is arguably the most powerful second-generation functional genomics technology currently available [Klemic]. Its high robustness, specificity, and efficacy in silencing targeted genes suggests its potential to father the development of a whole new class of drugs for an incredibly broad range of diseases. Before this can happen, however, significant challenges with respect to short interfering RNA (siRNA) delivery and targeting must be overcome.

One way to traverse the cell membrane and access the cell\'s interior is by temporarily increasing the permeability of the cell membrane. This can be accomplished via electroporation, a technique which uses high electric fields to induce structural rearrangements of the cell membrane. Pores result when the transmembrane potential exceeds the dielectric breakdown voltage of the membrane (0.2-1.5V) [Weaver]. Polar substances otherwise impermeant to the plasma membrane (such as dyes, drugs, DNA, proteins, peptides, and amino acids) can thus be introduced into the cell.

In the early 1980s, Eberhard Neumann et al. demonstrated the feasibility of electroporation for delivering DNA to a population of mammalian cells [Lundqvist]. Since then, this method of bulk electroporation has become a standard technique routinely used to simultaneously transfect millions of cells in culture. Most commercially available electroporation systems still use Neumann\'s approach without too much variation. Bulk electroporation requires very high voltages (kVolts) and has little control over the permeabilization of individual cells, resulting in suboptimal parameters. Moreover, because different cell types require different electric field parameters to electroporate, the system has to be calibrated to determine appropriate pulse conditions a priori without any real-time control. Reversible electroporation, in which the pores can reseal, is therefore difficult [Chang, D. C.]. As a result, most commercial systems focus on improving buffer solutions to improve cell viability. Examples of commercial electroporation platforms include the Gene Pulser Xcell™ Eukaryotic System (Bio-Rad Laboratories), BTX® HT 96 Well Electroporation System (BTX® Molecular Delivery Systems), Nucleofector™ 96-well Shuttle System (Amaxa Biosystems), and Axoporator 800A (Molecular Devices).

Single cell electroporation obviates many of the challenges associated with bulk electroporation but is less common. Lundqvist et al first demonstrated single cell electroporation using carbon fiber microelectrodes in 1998 [Lundqvist]. To induce electroporation, they placed microelectrodes 2-5 microns away from adherent progenitor cells. Other single cell electroporation techniques developed since include: electrolyte-filled capillaries [Nolkrantz (Electroporation)], micropipettes [Hass, Rae], and chips [Huang]. For successful single cell electroporation, the cell must either be isolated or the electric field well focused to target a particular cell [Nolkrantz (Functional Screening)]. Currently single cell electroporation is performed using laborious manual setups.

Contact adhesion between cells and surfaces, both inside and outside organisms, are central to a large number of biological phenomena. Some examples are blood clotting, tissue repair, immune and inflammatory response, bacterial infections, and cancer progression. A widely used method to quantify cell adhesion has been the application of a range of shear forces in flow chambers. The same methods are used to determine the cellular response to shear stress through mechanotransduction pathways.

The bulk of this type of research is currently performed using macroscopic laminar flow chambers. Current practice suffers from limited throughput, cumbersome apparatus assembly, experiment failure (i.e. by bubble introduction), and a limited range of applicable shear forces.

Thus, there remains a considerable need for alternative designs of microfluidic devices for manipulation of cells to support flow cytometry, cell ion-channel electrophysiology, single cell electroporation, controlled shear force in vivo-simulating cell culture and related technologies. The present invention satisfies these needs and provides related advantages as well.

SUMMARY

OF THE INVENTION

Aspects of the invention may include one or more of the following advantageous features: The system described herein can be useful to investigate dynamic processes at the cellular systems level and further to leverage related findings to engineer therapeutically useful cells for molecular medicine. Such cell uses can include but are not limited to applications to combat infectious diseases, neurodegenerative diseases, genetic disorders, and cancer.

In general, in one aspect, the invention features a microfluidic device for analysis of individual particles in a suspension including a substrate with one or more microfluidic channels adapted for individual addressability of particles, said substrate coupled to a plate with an array of apertures. In one embodiment the plate is a multi-well microplate. In another embodiment the multi-well microplate is selected from the group consisting of a 24-well, 96-well, 384-well, and 1536-well microplate.

In one embodiment the substrate is selected from the group consisting of a polymer, glass and quartz. In one embodiment the polymer is polydimethylsiloxane (PDMS). In another embodiment the substrate is integrated into the microplate such the microfluidic channels face upward. In a further embodiment the substrate that microchannels are molded into is continuous without punched holes.

In one embodiment the microfluidic device further includes an intermediate substrate disposed between the substrate and the plate.

In another embodiment the particles are selected from the group consisting of beads, cells, bacterial cells, vesicles, Oocytes, collection of cells and embryos.

In general, in another aspect, the invention features a microfluidic system for the analysis of particle suspensions including a microfluidic layer comprising at least one microscale flow channel adapted to contain particle suspensions, and an observation area for imaging the particle suspensions; a structure comprising one or more reservoirs in fluid communication with the microfluidic layer, wherein the reservoirs of the structure are in fluid communication with the microflow and main flow channels of the microfluidic layer; an interface detachably connectable to the microfluidic layer, wherein the interface controls fluid flow and pressure to one or more reservoir, thereby controlling pressure delivery to each microscale flow channel.

In one embodiment the system further includes a gasket between the interface and the device wherein the gasket is adapted to support a pressure seal between the interface and the device. In a related embodiment the pressure seal between the interface and the device includes a mechanical pressure seal between the gasket and the device. In one embodiment the pressure seal between the interface and the device is a negative pressure seal between the gasket and the device.

In a particular embodiment the interface includes manual regulators to regulate the pressure delivery to the one or more reservoirs. In one embodiment the manual regulators include manual toggle valves. In a related embodiment the interface includes electronically controlled regulators to regulate the pressure delivery to the one or more reservoirs. In another embodiment the electronically controlled regulators include electronically controlled valves.

Implementations of the invention can also include one or more of the following features. In one embodiment the particles are selected from the group consisting of beads, cells, bacterial cells, vesicles, Oocytes, collection of cells and embryos. In another embodiment the main flow channel is substantially 100-2000 um in width and substantially 5-200 um in depth. In a further embodiment a plurality of microscale channels are in fluid communication with one reservoir of the structure. In another embodiment the main flow channel is connected to a plurality of reservoirs of the structure, and wherein microscale channels terminate in the reservoirs.

In general, in another aspect, the invention features a method for analyzing a plurality of individual particles in suspension including repeatedly introducing pluralities of particles into a microfluidic chamber via flow; repeatedly acquiring images of said particles; and analyzing said images to characterize the particle population.

Implementations of the invention can include one or more of the following features. In one embodiment the microfluidic chamber comprises a section of a microfluidic channel. In another embodiment the flow comprises an adjustable flow velocity and wherein the flow velocity is adjustable between a flow velocity greater than 100 um/s and a flow velocity less than 1 um/s. In a further embodiment image acquisition occurs during periods of flow velocity less than 1 um/s. In a related embodiment analyzing images includes creating a set of individual particle images. In one embodiment characterizing the particle population includes determining a particle population characteristic selected from the group consisting of total particle counts, particle density in suspension, and particle size distribution. In another embodiment the particles are selected from the group consisting of beads, cells, bacteria, vesicles, Oocytes, and embryos.

In one embodiment characterizing the particle population includes characterization selected from the group consisting of determining fluorescent intensity, determining fluorescent marker distribution within the body or periphery of the particle, and classification of particles based on fluorescent intensity and/or fluorescent marker distribution and/or fluorescence lifetime. In a related embodiment images are acquired utilizing a method selected from the group consisting of optical microscopy, fluorescence microscopy, phase contrast microscopy, and confocal microscopy. In one embodiment analysis of the images comprises automatic particle recognition and storage of individual particle images.

In general, in another aspect, the invention features a system for performing particle imaging, counting, characterization, and classification including a microfluidic device containing a chamber adapted and arranged for simultaneous imaging of a plurality of particles; a flow actuation system in fluid communication with the microfluidic device that can introduce a population of particles into the chamber; an image acquisition system positionable for imaging the chamber; and an image analysis system in communication with the image acquisition system.

Implementations of the invention can include one or more of the following features. In one embodiment particles are selected from the group consisting of beads, cells, bacterial cells, vesicles, oocytes, collection of cells and embryos. In another embodiment the chamber includes a section of a microfluidic channel. In a further embodiment the microfluidic channel includes part of a microfluidic network. In one embodiment the microfluidic channel includes an inlet and outlet each in fluid communication with one or more reservoir. In another embodiment the reservoirs are disposed in a standard well plate format, selected from the group consisting of 6-well, 24-well, 96-well, 384-well, and 1536-well plates.

In a particular embodiment the flow actuation system is adapted to provide a flow velocity adjustable between a flow velocity greater than 100 um/s and a flow velocity less than 1 um/s. In one embodiment the flow actuation system includes a pressure application apparatus. In another embodiment the microfluidic channel includes an inlet and an outlet and the pressure application apparatus is adapted to apply a differential pressure to the inlet and outlet of the microfluidic channel. In one embodiment the flow actuation system includes an electrokinetic flow apparatus.

In one embodiment the image acquisition system includes a microscope and a CCD camera. In a related embodiment the image acquisition system includes a microscope objective and a CCD camera, mounted in an enclosure. In a particular embodiment the enclosure includes a microscope chassis.

In a further embodiment the image analysis system includes a microprocessor and a software application. In a related embodiment the software application is adapted to identify individual particles among the imaged particles. In another embodiment the software application is adapted to measure the size and morphological parameters of identified particles. In yet another embodiment the software application is adapted to classify imaged particles based on the size and morphological parameters of imaged particles. In one embodiment the software application is adapted to measure a fluorescence intensity and fluorescent distribution inside a perimeter of each identified particle. In another embodiment the software application is adapted to classify imaged particles based on the measurement of fluorescence intensity and fluorescent distribution inside the perimeter of each identified particle.

In general, in another aspect, the invention features a microfluidic system for sorting individual particles based on optical observation including a microfluidic layer with at least two microscale channels intersecting with a main flow channel; a multi-well structure in fluid communication with the microfluidic layer, with the wells of said multi-well structure in fluid communication with the channels of the microfluidic layer; an interface which is removably coupled to the microfluidic device which controls the flow of fluid in the microfluidic channels and can apply a positive or negative pressure to each of the wells of the multi-well plate, thereby applying positive or negative pressure to the microscale channels in the microfluidic layer; and a control system in optical and fluid communication with the microfluidic layer, and adapted for particle identification and selective pressure application depending on observed particle position.

Implementations of the invention can include one or more of the following features. In one embodiment the particle is selected from the group consisting of cells, bacteria, vesicles, oocytes, and embryos and particle identification includes an observation selected from the group consisting of observed particle size, particle morphology and particle surface marker.

In general, in another aspect, the invention features a microfluidic system including a structure including a plurality of open reservoirs; and a substrate including microfluidic channels on one side, said substrate coupled to the structure with the channel side facing the substrate, wherein the microfluidic channels are in alignment with the open reservoirs of the structure such that the reservoirs of the structure are in fluidic communication with the microfluidic channels. In one implementation the substrate includes one or more main flow channel, a plurality of trapping channels and a detection zone for viewing cells microscopically, wherein one or more reservoir is in fluid communication with one or more trapping channel, and wherein each trapping channel is in fluid communication with one or more main flow channel, and wherein the detection zone is adapted for viewing cells using an upright microscope or an inverted microscope.

In general, in another aspect, the invention features a microfluidic system for trapping individual particles in an array for analysis including a microfluidic layer including an array, at least two microscale channels and a main flow channel wherein the microscale channels intersect with the main flow channel and wherein the array is adapted to trap individual particles; a structure including a plurality of reservoirs coupled to the microfluidic layer, wherein the reservoirs of the structure are in fluid communication with the microscale and main flow channels of the microfluidic layer; and an interface detachably connectable to the microfluidic layer, wherein the interface controls fluid flow, and pressure to one or more reservoir, thereby controlling pressure delivery to each microscale channel.

Implementations of the invention can include one or more of the following features. In one embodiment the microscale channels are adapted to trap particles selected from the group consisting of cells, vesicles and oocytes. In another embodiment the microscale channels are substantially 0.5-10 um in width and substantially 0.5-10 um in depth. In yet another embodiment the main flow channel is substantially 100-200 um in width and substantially 20-100 um in depth. In one embodiment a plurality of microscale channels are in fluid communication with one reservoir of the structure. In another embodiment the main flow channel is connected to a plurality of reservoirs of the structure, wherein microscale channels terminate in the reservoirs.

In general, in another aspect, the invention features a method for analyzing a plurality of individual particles including disposing a suspension of particles into one or more reservoir of a microfluidic device including one or more reservoir and a plurality of intersecting channels in fluid communication with the one or more reservoir; immobilizing a plurality of individual particles at one or more junction of the plurality of intersecting channels; perfusing one or more compounds across the particles; and analyzing the plurality of individual particles.

Implementations of the invention can include one or more of the following features. In one embodiment the microfluidic device further includes a substantially planar substrate positioned below a plane of the bottom of the reservoirs, wherein the one or more junction of intersecting channels and the immobilized particles are disposed within the planar substrate.

In one embodiment the particles are selected from the group consisting of cells, vesicles and oocytes. In another embodiment analyzing includes measuring properties from one or more of the immobilized individual particles. In yet another embodiment compounds are perfused inside the intersecting channels. In a further embodiment the intersecting channels are part of a microfluidic network of channels.

In another embodiment analyzing comprises taking measurements selected from the group consisting of whole cell voltage clamping, whole cell current clamping, and patch clamping. In a related embodiment analyzing includes taking measurements selected from the group consisting of optical microscopy, fluorescence microscopy, phase contrast microscopy, and confocal microscopy. In another embodiment the compounds are selected from the group consisting of biomolecules, small molecules, proteins, enzymes, genetic material, biomarkers, and dyes.

In general, in another aspect, the invention features a system for single particle analysis the system comprising a microfluidic device comprising one or more reservoirs and adapted for holding and manipulating particles and compounds; and an interface adapted to provide pressure to the one or more reservoirs, wherein the interface is detachably coupled to the microfluidic device. In one implementation the microfluidic device is integrated into a microplate format selected from the group consisting of a 24-well, 96-well, 384-well, and 1536-well microplate, or a section thereof.

In general, in one aspect, the invention features a system for conducting patch clamp measurements on an array of immobilized particles, wherein the distance between immobilized particles is substantially below 0.1 mm. In one implementation the particles are selected from the group consisting of cells, vesicles and oocytes. In one embodiment the particles are cells. In a related embodiment a plurality of the cells can be simultaneously observed microscopically. In another embodiment the system further includes one or more compound streams in fluid communication with the immobilized cells wherein a plurality of the cells are exposed to the same compound stream.

In general, in another aspect, the invention features a system for conducting patch clamp experiments including a microfluidic device with at least two intersecting channels and a detection zone; an interface detachably coupled to the microfluidic device, wherein the interface is adapted for moving material within the microfluidic device; and an electrode array electrically and fluidically in communication with the microfluidic device.

Implementations of the invention can include one or more of the following features. In one implementation the microfluidic device is integrated into a structure including a plurality of reservoirs. In one embodiment the structure includes a microplate, the microfluidic device is integrated into the microplate and the microplate is selected from the group consisting of a 24-well, 96-well, 384-well, and a 1536-well microplate. In another embodiment the interface is coupled to a section of the microfluidic device.



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stats Patent Info
Application #
US 20120264134 A1
Publish Date
10/18/2012
Document #
13454849
File Date
04/24/2012
USPTO Class
435/613
Other USPTO Classes
435 29, 435/61, 435/721, 4352887, 4352871, 4352872
International Class
/
Drawings
51


Electrophysiology


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