Microwell arrays with nanoholes

A device for conducting parallel analysis or manipulation of multiple cells or biomolecules is disclosed. In one embodiment, the device comprises a silicon chip with a microwell, and at least one membrane suspended at the bottom opening of the microwell. The suspended portion of the membrane defines a nanohole that provides access to the material on the other side of the membrane.

TECHNICAL FIELD

A device for conducting parallel analysis or manipulation of multiple cells or biomolecules is disclosed. The device may be used for a number of applications such as parallel patch-clamping, precision delivery of biochemical factors to cells, high-throughput screening for drug discovery, immunoisolation of cells, cellular and biomolecular separation, and protein and DNA sequencing.

BACKGROUND OF THE INVENTION

One of the biggest challenges in cell biology, as well as in neuroscience, is being able to do parallel analysis on multiple individual cells. This stems from the fact that most of the current cellular analysis tools and methods are not chemically sensitive enough for or physically capable of obtaining measurements from single cells. They also are not suitable (e.g. too bulky) to be implemented into automated systems for parallel analysis and require relatively extensive human labor. Furthermore, cell-based assays are becoming an essential step in high-throughput screening (HTS)-based drug discovery processes, increasing the demand for ever faster cell analysis tools. One solution of this problem is miniaturization of the cell analysis tools and methods using the state-of-the-art micro- and nanofabrication techniques. The device presented here is cheaper and more scalable than the conventional devices and has much higher throughput. It can be automated and do parallel analysis on multiple cells simultaneously. In addition, it requires much less expertise and operator time. Because of its flexible design, it can be used for a diverse range of applications such as parallel patch-clamping, precision delivery of biochemical factors to cells, high-throughput screening for drug discovery, immunoisolation of cells, cellular and biomolecular separation, and protein and DNA sequencing.

SUMMARY OF THE INVENTION

The lab-on-a-chip device described here is a new generation cell culture tool that allows the interrogation of large number of single cells simultaneously. The device enables parallel electrophysiological measurements, such as measuring ion currents through ion channels residing in the plasma membrane, as well as delivery of key biochemical factors to multiple cells with a very high precision. To achieve this, an array of very small holes is created on a thin transparent membrane of silicon nitride or silicon oxide that is suspended on an array of wells through a silicon wafer. A microfluidic channel network, made of poly(dimethylsiloxane) or another appropriate polymer, on the membrane side of the wafer allows individual addressing of the holes. Because the holes provide the sole fluidic and electrical connection between the microfluidic network on one side and the wells on the other side, very low-noise electrical measurements and well-controlled high-resolution delivery of biomolecules to cells can be achieved. Individual cells are cultured inside each well on top of or a certain distance away from the hole, depending on the application. The cells can also be cultured on the membrane side of the device, and the fluidic delivery can be done from the well-side. In addition, both sides of the device can be separately addressed via microfluidic channels. The device is useful for a number of other applications, such as high-throughput screening for drug discovery, immunoisolation of cells, cell filtration and separation, biomolecular separation, and protein and DNA sequencing.

Additional aspects and advantages of this invention will be apparent from the following detailed description of preferred embodiments, which proceeds with reference to the accompanying drawings.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In one embodiment, the device is a lab-on-a-chip and is capable of automated, parallel, patch-clamp analysis of the effect of drug compounds on ion channels. The device can perform a high number of simultaneous patch-clamp evaluations with low drug compound and reagent consumption. For example, the device may have over600patch clamp units. The device is capable of performing independent modulation of chemical and electrical environment in each patch clamp unit. In the device, cells are automatically guided to the holes on the patch-clamp units by the application of suction from the opposite side of the hole. Each patch clamp unit may be individually addressed and is fluidically and electrically isolated from the rest of the units on the chip. The user can alter the electrical and chemical conditions of the solutions on either side of the cell. The device is further capable of rapid fluid exchange, which allows the study of ligand-gated ion channels.

FIG. 1is an image showing the top view of an embodiment of the device. The device comprises sixteen patch clamp units, each unit comprising a microwell60and a microchannel80. Also seen inFIG. 1are alignment marks40. Alignment marks40assist in the manufacture of the device, as discussed in more detail below.

FIG. 2is a cross-sectional view of an embodiment of the device adapted for patch clamping. The device comprises a substrate10that defines at least one microwell60. Each microwell60comprises a top opening64so that the microwell can receive fluid. Each microwell further comprises a bottom opening62. A silicon nitride membrane20surrounds substrate10, except at top openings64, where membrane20defines a window24. At bottom opening62of each microwell60, a portion28of membrane20is suspended and defines a nanohole22. As a result, fluid placed in the microchannel is in fluid communication with the fluid in the microwell via the nanohole.

In the embodiment shown inFIG. 2, a microfluidic channel layer70comprises microchannels80with microchannel openings82. Layer70is positioned below the substrate or wafer so that each microwell60and corresponding nanohole22is oriented over a microchannel opening82. In the embodiment shown inFIG. 2, microchannels80are enclosed within channel layer70except at openings82. In another embodiment (not shown) the microchannels may be exposed to the silicon nitride layer along their entire length.

In one embodiment, channel layer70comprises poly(dimethylsiloxane) (“PDMS”), a polymer that provides insulating properties for use in electrophysiological measurements. However, any polymer may be used that is capable of sealing to the silicon nitride layer, forming microchannels, and, for applications involving electricity, capable of providing insulation. For example, polyimide may be used for the channel layer.

Depending on the application and the desired results, a cell or biomolecule may be positioned on either side of the suspended membrane. For example, for patch clamping a cell may be placed on the microwell side of the suspended membrane.

The size of the nanohole can vary depending on the application. For example, for delivery of biomolecules to a cell, the nanoholes may range in size from about 1 to 100 microns. For patch-clamp experiments, nanoholes range in size from 0.5 micron to about 3 microns. For DNA or peptide sequencing, nanoholes range in size from about 1 to about 4 nanometers.

The embodiment shown inFIG. 2is adapted for patch clamping on multiple individual cells. As shown inFIG. 2, each microchannel80has a portion82with an area below microwell bottom opening62. Portion82is smaller than the area defined by the perimeter of microwell bottom opening62. This configuration enables the microchannel to contain a fluid such that the fluid contacts only the suspended portion of the membrane. Stated otherwise, the configuration of the membrane, the microchannel and the microwell bottom opening prevents fluid contained in the microchannel from contacting a portion of the membrane that is below the substrate.

The following list provides some of the device's potential applications. The device may be used to do anything the conventional methods and devices do, but more accurately, more quickly, with less cost and less expertise. The first two of these applications are illustrated inFIGS. 3-6.Parallel electrophysiology on multiple individual cells (e.g. parallel patch-clamping)Precision delivery of biochemical factors to cellsHigh-throughput screening (HTS) for drug discoveryImmunoisolation of cellsCellular filtration and separationBiomolecular separationProtein and DNA sequencing

Conventional patch-clamping is a common approach for evaluating the effect of compound on ion channels. Traditionally, patch-clamping involves sucking a portion of the cell membrane containing the ion channel of interest into the mouth of a pipette with about 1 micrometer internal diameter. A voltage is applied across the ion channel by placing electrodes in the electrolyte solution within the micropipette and the cell bath solution. Prospective drug compounds are then applied to the micropipette solution or the cell bath solution and the corresponding effects on ion channel function are evaluated.

FIG. 3Aillustrates traditional patch clamping, which requires an operator to guide the tip of a micropipette100to a cell110.FIG. 3Bshows the operation of an embodiment of the inventive device. The device comprises a silicon wafer10having a silicon nitride membrane20.FIG. 3Bshows an expanded view of an embodiment of the invention designed for patch clamping. As shown inFIG. 3B, a nanohole22in membrane20is aligned with microwell60. The device is capable of automatically guiding a cell110to nanohole22on the patch-clamp units when suction is applied to the opposite side of the nanohole.

FIG. 4shows the use of the embodiment shown inFIGS. 1-2for planar parallel patch-clamping. The electrical isolation of the silicon-backed sections of the silicon nitride membrane from the fluid (electrolyte) inside the microchannels enables the device to provide measurements with a minimal amount of noise. For example, meaningful measurements are generally taken in picoamperes (10−12amperes).

FIGS. 5-6illustrate precision delivery of biochemical factors to cells. A different biomolecule solution may be delivered to each cell in the microwell60via the microchannels80and nanoholes22. The device is adapted to achieve delivery of factors to a small area of the cell membrane. The device can deliver factors in a parallel fashion to multiple individual cells (i.e. high-throughput) and can be automated to save operational costs. In addition, use of the device for such delivery requires a relatively low level of skill and low levels of fluids and reagents.

The device may be used for efficient and reliable high-throughput screening for drug discovery. Screening may be conducted by patch clamping, as described above.

Other applications for the device include cellular filtration and separation. An embodiment for filtration comprises a suspended membrane defining multiple holes with precise diameters. The multiple holes are capable of action as filters. Some small cells may be allowed through the holes, while other, larger cells cannot pass through the holes. The separation can also be based on cell motility, where cells migrating on the surface of the suspended membrane or in the liquid medium travel to the other side through the holes. This application may be useful for separating healthy and motile spermatozoids from dead ones.

The device may be used for biomolecular separation. The holes on the membrane are fabricated small enough to permit proteins and biochemical factors up to a certain size to pass through the membrane. Separation can be driven by any suitable means, such as diffusion, pressure, electrical or magnetic fields and centrifugation.

The device may also be useful for protein and DNA sequencing. For this application, the nanoholes in the membrane may measure about 1 nanometer to about 4 nanometers, which is roughly the diameter range of single stranded DNA and proteins. To conduct sequencing, electrical current is passed through the device. As the molecules are forced through the hole, the electrical current that can flow through the hole changes in relation to size of each base or amino acid. The bases of DNA or amino acid of a peptide may then be determined from their characteristic current signatures.

The fabrication process of the device is described in connection withFIGS. 7-14.

FIGS. 7A-7Bshow perspective and cross-sectional views of a standard or test grade silicon wafer10about 0.4 mm thick and with <100> crystal orientation. The wafer thickness may vary depending on the desired well size. Silicon, when etched, forms angled walls. As a result, the microwells have angled walls, as shown inFIG. 1. Because of this angle, a thinner wafer allows the microwells to be positioned closer together than a wafer of greater thickness.

A thin (200-2000 nm) layer20of low-stress silicon nitride is deposited on wafer10, as shown inFIGS. 8A-8B. Layer20may be deposited by any conventional method, such as low pressure chemical vapor deposition (“LPCVD”) or plasma enhanced chemical vapor deposition (“PECVD”).

After a layer of silicon nitride is deposited on the wafer, a layer of metal30is deposited on the top side of the wafer. Metal layer30is etched to create alignment marks used to orient the wafer, as discussed in more detail below.FIGS. 9A-9Billustrate this metal evaporation step. In a sputter or thermal evaporation system, a 100 nm layer30of gold (Au) is deposited on front side12of wafer10with a 10 nm layer of chromium (Cr) or titanium-tungsten alloy (TiW) (not shown) as the adhesion layer between silicon nitride and gold layers. Alternatively, layer30could be any metal that can be patterned by etching or dissolution. For example, a layer of chromium or titanium-tungsten alloy could be used instead of the gold layer.

After deposition of the silicon nitride and metal layers, alignment marks40are made in the metal layer, as illustrated inFIGS. 10A-10B. A polymer that can be patterned via exposure to light, such as positive photoresist AZ1512, is used with standard photolithographic techniques to create photoresist islands in the shape of the desired alignment marks on the gold surface. These photoresist islands protect the underlying Au and Cr or TiW during the subsequent metal etch processes, resulting in alignment marks with the desired shape. Any pattern may be used that enables the features on the top and bottom of the wafer to be aligned.FIG. 10Cshows one set of Au alignment marks created by this process on the silicon nitride layer.

Following metal deposition, nanoholes are created in membrane20by electron-beam followed by reactive ion etching (RIE), shown inFIGS. 11A-11B. Using electron beam lithography, an array of nanoholes are formed in a poly(methylmethacrylate) (PMMA) film90in registry with the alignment marks.FIG. 11Cshows a scanning electron microscope image of an array of 500 nm-diameter holes in a 0.75 μm-thick PMMA film90. The holes can be fabricated with a diameter ranging from tens of nanometers to tens of micrometers, depending on the application. Using the PMMA layer as the etch mask, the array of nanoholes is transferred into the silicon nitride membrane20by any suitable process. For example, the nanoholes may be created in the silicon nitride membrane also by photolithography followed by any dry plasma etching technique that is capable of selectively etching the silicon nitride. PMMA is then removed in an acetone bath.

Photolithography may be used to create nanoholes as small as about 500 nanometers. Smaller size nanoholes may be formed by electron beam lithography down to 50 nanometers. Nanohole sizes may be further shrunk by deposition of silicon nitride or silicon dioxide in the nanoholes.FIG. 11Dis an SEM image of a 500 nm-diameter nanohole in a silicon nitride membrane.FIG. 11Eis an SEM image of a 1000 nm-diameter nanohole in a silicon nitride membrane.FIG. 11Fis another SEM image of a 500 nm-diameter nanohole in a silicon nitride membrane.FIG. 11Gis an SEM image of a less than 200 nm-diameter nanohole in a silicon nitride membrane.

FIGS. 12A-12BandFIGS. 13A-13Billustrate the next steps, etching of microwells windows24in silicon nitride layer20and anisotropic etching of microwells60in wafer10. Square etch-windows24in the silicon nitride layer are formed on the backside of the wafer in registry with the alignment marks on the front side using standard photolithographic techniques and RIE, as shown inFIGS. 12A-12B. Only areas defined by window24will be etched away to create microwells. An aligner/exposer system with backside alignment capability is employed during the photolithography process. The wafer is anisotropically etched through the etch windows in a potassium hydroxide (KOH) bath (24.5% w/w, 80° C.) until the other side is reached, as shown inFIGS. 13A-13B.FIG. 13Cis a top view of an array of microwells etched into a wafer with the inset magnifying one of these wells. This process suspends an array of silicon nitride membranes with a nanohole at the center of each.FIG. 13Dis an image of a membrane taken from the back side (the microwell side) of the wafer.

FIG. 14Aillustrates the step of microchannel assembly. A poly(dimethylsiloxane) (PDMS) channel layer70containing a network of microchannels80is assembled onto the front side of the wafer in registry with the suspended silicon nitride membranes. The microchannels are enclosed except for openings below the microwells at the suspended silicon nitride. Any polymer capable of sealing to the silicon nitride, such as polyimide, may be used instead of PDMS. PDMS layer70may be created separately and then sealed to the silicon wafer or may be applied directly. To get a good seal and adhesion between the silicon nitride and PDMS, both PDMS microchannels and wafer are treated in oxygen plasma to activate their surfaces. This allows a robust chemical bonding between them. Alternatively, a photocurable polymer may be directly patterned onto the silicon nitride layer by photolithography to form the channel layer.FIG. 14illustrates the relative sizes of microchannel80, nanohole22and bottom opening of microwell62.

Modifications to the embodiments of the invention described above include the following:

Another wafer substrate such glass, quartz or sapphire can be used instead of silicon, and the fabrication process can be modified to accommodate this substrate of choice.

Silicon dioxide can be deposited onto the wafer and used as the membrane layer instead of silicon nitride. Other materials which are suitable for membrane materials include transparent polymers. These membrane materials can be deposited, grown or coated to form the appropriate membrane layer.

Another metal can be employed to create the alignment marks.

The number of membrane windows and microwells in the array can be increased or decreased depending on the needs of the application.

For creating the holes on the membrane, the electron beam lithography process can be substituted by a photolithographic process with a high-resolution chrome photomask. This will further lower the production costs of the device.

In place of PDMS, the microfluidic network can be fabricated from another suitable polymer.

Instead of assembling the microchannel network onto the wafer, the network can be created directly on the wafer surface by using a light-curable polymer photoresist (such as SU-8 from MicroChem Corp.) by standard photolithography.

The electrodes necessary for the electrophysiological applications of this device can be formed on the wafer surface by conventional photolithographic and metal-etch techniques. This will eliminate the need for external electrodes and increase the ease of use of the device, while decreasing the cost of operation.

EXAMPLES

FIG. 15Ais a cross-sectional view of a device comprising a silicon wafer10defining a microwell60. A PDMS layer70with a microchannel80is positioned with the microchannel aligned with a microwell60. Membrane layer20does not have a nanohole below microwell60.FIG. 15Bis a top view of the device shown inFIG. 15A. Test experiments were conducted using this device to determine whether it provides results comparable to a traditional patch-clamp set-ups. The microwells and microchannels were filled with electrolyte fluid. A square-wave voltage pulse was applied between microwell and the microchannel. Electrical resistance and capacitance were measured. The results of the experiments are shown inFIG. 15C. In the absence of a nanohole the current did not flow, showing that the suspended silicon nitride layer provides a good insulation.

FIG. 16Ais a cross-sectional view of a device similar to the device described in connection withFIGS. 15A-15Bexcept that membrane20defines a 1 μm nanohole22between microwell60and microchannel80.FIG. 16Bis a top view of the device shown inFIG. 16A. Electrical characterization of this setup was conducted in accordance with the protocol described above for Example 1. The results are shown atFIG. 16C. The resistance and capacitance values were comparable to micropipette resistance with a tip diameter of 1 μm.

FIG. 17Ais a cross-sectional view of a device similar to the device described in connection withFIGS. 16A-Bexcept that microchannel80is misaligned with microwell60.FIG. 17Bis a top view of the device shown inFIG. 17A. Electrical characterization of this setup was conducted in accordance with the protocol described above for Example 1. The results are shown atFIG. 17C. The results indicate that there was cross-talk or interference between the two electrodes of the amplifier through the silicon-backed (unsuspended) silicon nitride layer and the bulk silicon.

These examples illustrate that the device shown inFIGS. 16A-16Bis optimized for patch-clamping applications.