CA2817775A1

CA2817775A1 - An integrated microfluidic device for single-cell isolation, cell lysis and nucleic acid extraction

Info

Publication number: CA2817775A1
Application number: CA2817775A
Authority: CA
Inventors: Unknown
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-05-29
Filing date: 2013-05-29
Publication date: 2014-11-29

Abstract

A microfluidic platform which integrates a cell trapping barrier and nanochannel array for genomic/epigenomic analysis of single DNA molecules. The system consists of a fused silica "chip" with the aforementioned features and a pressure-resistant plastic "chuck" that connects to a fluorescence microscope and is used to operate the chip using fluid pressure and fluid pumps. A cell sample is input directly into the device where it is captured and burst. Nucleic acids are collected in the nanofluidic channel array where experiment techniques such as the "barcoding" analysis is performed.

Description

10021 This invention is to solve the problem that single-cell genome/epigenome analysis is slow, expensive, and prone to error and contamination. It provides a method for integrating single-cell isolation, cell lysis and nucleic acids extraction.
This is achieved by designing microchannels and nanochannels with distinct geometric designs that allow one to obtain the desired cell from a number of cells quickly and accurately, break open the cell membrane and nuclear membrane, release the nucleic acids, and direct the nucleic acids into the nanochannel array.

10041 This invention relates to a fused silica microfluidics chip (hereinafter referred to as a "chip") to implement single-cell isolation, cell lysis, and nucleic acids extraction. Further, it relates to a method utilizing liquid pumping and fluid pressure both together to transfer cells during a sorting operation, and relates to micrometer-scale and nanometer-scale designs for single-cell isolation, cell lysis, and nucleic acids extraction.
10051 Various embodiments of the chip include sample wells 5, microchannels (1,2,3 and 4), T-junction 6, cell trap 7, barrier slit 8, buffer zone 9, and nanochannel array 10. The sample wells allow the user's cell sample to be deposited into the chip.
The size of the wells allow the user to deposit the sample in microliter amounts.
Various embodiments of the invention allow users to deposit the cells into one or many wells. In various embodiments, users can use an application-specific cell buffer, including an application-specific fluorescent dye for labeling the nucleic acids. In various embodiments, the cells are carried through the microchannels by liquids towards the T-junction. At the T-junction, the invention allows users to select a desired cell based on visual or biochemical characteristics. In various embodiments, users can capture the selected cell in the cell trap by applying certain magnitudes of hydrostatic pressure to the correct combination of microchannels.
10061 Various embodiments of the invention allow users to select the desired cell by size according to the depth of the cell trap. Shallower cell traps will be able to capture only smaller cells. The size of the captured cell is determined by the depth of the cell trap in the following way: the diameter of the desired cell to be captured must be larger than the depth of the cell trap. In various embodiments, a cell lysis buffer is driven using liquid pumps towards the cell trap to solubilize the cell membrane and the nuclear membrane and facilitate the release of nucleic acids. The cell lysis buffer is driven into the device through a dedicated sample well. In various embodiments, users can complete cell lysis by applying fluid pressure, using liquid pumps, to the cell lysis buffer sample well and microchannel. Maintaining fluid pressure in the previously described configuration and direction will liberate the cell's nucleic acids and pump them into the barrier slit. Various embodiments of the barrier slit will contain micrometer-scale posts, micrometer-scale pits, nanochannels, or any combination thereof for the purpose of trapping and sorting nucleic acid molecules.
The embodiment described henceforth has a barrier slit consisting of an array of micrometer-scale posts.
10071 Various embodiments of the device will include an array of nanochannels for optical nucleic acids analysis. The nucleic acids are driven into the nanochannel array by standard electrophoresis techniques. The nucleic acids uncoil and become linearized in the nanochannels. In various embodiments, temperature or chemical treatment of the nucleic acids inside the nanochannels will induce the dye to become free of the nucleic acid, depending principally on the underlying genetic sequence. In various embodiments, the induced genetic "barcode" can be captured using a fluorescent microscope and saved as an image. In various embodiments, the image files can be analyzed using dedicated software. Further applications of the nanochannel array include but are not limited to techniques that rely on optical measurement of long strands of single DNA molecules.

10091 Figure 1. Fig. 1 is a schematic top view of the fused silica chip.
100101 Figure 2. Fig. 2 is an enlarged schematic view of the dotted area in Fig. 1.
100111 Figure 3. Fig. 3 shows the first step of the method for sorting single cells according to the present invention.
100121 Figure 4. Fig. 4 shows the third step of the method for sorting single cells according to the present invention.
[00131 Figure 5. Fig. 5 shows the fourth step of the method for sorting single cells according to the present invention.
100141 Figure 6. Fig. 6 shows the fifth step of the method for sorting single cells according to the present invention.
100151 Figure 7. Fig. 7 shows the sixth step of the method for sorting single cells according to the present invention.
100161 Figure 8. Fig. 8 is a schematic view showing the device used for the method for cell lysis according to the present invention.
100171 Figure 9. Fig. 9 is a schematic view of the buffer zone and the nanochannel array.
100181 Figure 10. Fig. 10 is a Scanning Electron Microscope photo of the nanochannel array and post array.

100191 Figure 11. Fig. 11 is a photo view of the experiment setup.

IMPLEMENTATION
100211 Hereinafter, a single embodiment of a chip and method for single cell sorting, cell lysis, and nucleic acid extraction will be described with reference to the drawings.
100221 The chip of this invention includes two cell traps 7, each with three sample wells 5, two cell supply microchannels 1 and 2, a cell sorting microchannel 4, a barrier slit 8, a buffer zone 9, and a cell exit microchannel 3. The two devices are arranged symmetrically, opposite in orientation, and connected at the center of the chip by the nanochannel array.
The cell supply microchannels for transferring cells delivered with a liquid from the syringe is a hollow microchannel about 30 um in depth and 90 urn in width. The cell supply microchannels are each connected with one sample well. The dimensions of the cell supply microchannels can subject to change according to specific needs such as the size of cell sample used.
100231 Similar to the cell supply microchannels, the cell sorting microchannel for sorting and transferring only the desired cells delivered with a liquid is a microchannel about 30 urn in depth, 90 um in width and 100 urn in length. The dimensions of the cell sorting microchannel can subject to change according to specific needs such as the cell selection accuracy desired. The known supply mechanism, such as a syringe pump and a syringe, for supplying the liquid in the microchannels can be applied to the above-described channels via the respective sample wells.

[0024] The cell sorting microchannel intersects with the cell supply microchannels at the T-junction. In this embodiment of the invention, their crossing angle is degrees. The angle of crossing between the cell sorting microchannel and the cell supply microchannels can subject to adjustments according to specific needs such as the cell selection accuracy desired.
[0025] The cell trap is the interface between the cell sorting microchannel and the barrier slit.
[0026] The barrier slit is 1 urn in depth, 40 um in width, and 200 um in length.
100271 The barrier slit contains post array with diameters of 2 urn arranged diagonally and in an "upper triangular" fashion and are spaced at 2 urn intervals. Each row of posts is stacked on the previous row, but displaced to the opposite side of the cell sorting microchannel by 10 urn, forming a slanted arrangement, as illustrated in Figure 8.
[0028] The buffer zone is similar to the barrier slit. The buffer zone is 1 urn in depth, 40 urn in width and 150 um in length. The buffer zone contains post array arranged diagonally with diameters of 2 urn, 1 urn and 0.5 um spaced at 2 urn, 1 urn and 0.5 um respectively arranged in descending order of diameters and intervals towards the nanochannel array, as illustrated in Figure 9.
[0029] The nanochannels in the nanochannel array are 120 am in width and 200 am in depth, arranged in parallel with intervals of 380 urn.
[0030] Next, the cell sorting method of this invention will be explained.
100311 Fig. 11 is a photo view showing one example of a device used in the cell sorting method of this invention. The device consists of an inverted fluorescence microscope combined with a halogen light source and so on, and syringes with tubings connected to the chuck 12, the loading platform necessary for chip performance.
100321 The inverted fluorescence microscope 17 includes an electronic stage 13 capable of moving along an X-Y axis and supporting the chuck that holds the chip, an objective lens placed directly below the electric stage to focus a light from the light source and guide the light to the chip, a halogen lamp that sends the light to the chip, an electric shutter placed between the light source and the chip to control a quantity of light from the halogen lamp to the chip, and an imaging device such as CCD
camera which captures a transmission image of a visible light from the halogen lamp passing the chip.
100331 5m1 syringes 14 with male luer-lok fittings are connected to 1/4 inch-diameter teflon tubings 16. Two-way and three-way valves 15 are placed between the syringes and the tubings. The tubing is connected on the other end to the chuck by a plastic adapter. The size of the syringe is subject to changes according to the amount of cell samples provided.
100341 The chuck is a platform for chip mounting that is made of plastic.
Screw holes of specific sizes are drilled through from top to bottom according to specific needs.
Threadings are tapped in these holes for the purpose of screw anchoring. The threaded holes on the sides of the chuck are on one end connected to channels within the chuck that further connect to the holes on the top and bottom of the chuck, and on the other end cconnected to adaptors and then to tubings that connect to the valves and the syringes. The holes on the top and bottom of the chuck are aligned with the sample wells on the chip. The holes on the top are sealed with screws of corresponding sizes and plastic o-shaped rings for complete sealing. The channels within the chuck are designed to allow continuous fluid flow from the syringe through the tubings then the chuck and then to the sample wells on the chip. The design of the chuck can be adjusted according to specific needs such as the adaptation of a heating device.
100351 The method for sorting the desired cell from a number of cells, performing cell lysis and nucleic acid extraction according to the present invention are accomplished by using the above-described device as below.
100361 At first, the chip of the present invention described above is mounted on a chuck, which is fixed on the electric stage of an inverted fluorescence microscope.
The supply mechanism such as a syringe pump and syringes are prepared as well.
As shown in Fig. 11, the syringe containing the cell suspension is connected through tubings to the chuck and further to the sample well that connects to cell supply microchannel 1. The syringe containing the cell lysis buffer is connected through tubings to the chuck and further to the sample well that connects to cell supply microchannel 2, and the syringe containing the nucleic acid buffer is connected through tubings to the chuck and further to the sample well that connects to the cell exit microchannel.
100371 As the first step, the valves connected to the syringe that connects to cell supply microchannel 1 are adjusted to allow only fluid flow from that syringe into the cell supply microchannel 1. The valves connected to the syringe that connects to cell supply microchannel 2 are adjusted to allow only fluid flow from the cell supply microchannel 2 to the atmosphere. The supply mechanism sends liquid containing the cells to cell supply microchannel 1 and subsequently to cell supply microchannel 2, as illustrated in Figure 3.

[00381 As the second step, the liquid flowing in the cell supply microchannels 1 and 2 is observed to confirm that a desired cell is arrived in the T-junction, and then the liquid supply from the supply mechanism is stopped.
100391 As the third step, the valves connected to the syringe that connects to cell supply microchannel 1 and the valves connected to the syringe that connects to cell supply microchannel 2 are adjusted to allow only fluid flow from the cell supply microchannels to the atmosphere. Then, the liquid containing the cells is manipulated by adjusting the hydrostatic pressure difference between cell supply microchannel 1 and cell supply microchannel 2. Fine hydrostatic pressure adjustment is achieved by adjusting the displacement of the syringes from a reference horizontal plane.
When a desired cell arrived at the T-junction, the hydrostatic pressures at the two cell supply microchannels are adjusted to equal values to completely stop liquid movement at the T-junction. This is illustrated in Figure 4.
100401 As the fourth step, the desired cell is transferred to the cell sorting microchannel using hydrostatic pressure-driven flow, as illustrated in Figure 4. In this method, the valves connected to the syringe that connects to the cell exit microchannel are adjusted to allow only fluid flow from the cell exit channel to the atmosphere. Then, the syringe connected to the cell exit microchannel is adjusted to a lower vertical displacement with respect to the syringes connected to the cell supply microchannels. This creates a hydrostatic pressure difference across the barrier slit.
After transferring the desired cell to the cell sorting microchannel, the desired cell is further carried by the hydrostatic pressure-driven flow to the cell trap, as illustrated in Figure 5.
100411 As the fifth step, the valves connected to cell supply microchannel 2 are adjusted to only allow fluid flow from the syringe connected to cell supply microchannel 2 to cell supply microchannel 2. The supply mechanism sends liquid containing the lysis buffer to the cell supply microchannel 2 and subsequently to cell supply microchannel 1 to displace liquids containing cell samples in cell supply microchannel 1. This step is illustrated by Figure 6.
100421 As the sixth step, the valves connected to cell supply microchannel 1 are adjusted to impede fluid flow to the syringe connected to cell supply microchannel 1 or the atmosphere. The valves connected to cell supply microchannel 2 are adjusted to allow only fluid flow from the syringe connected to cell supply microchannel 2 to cell supply microchannel 2, while the supply mechanism sends liquid containing the lysis buffer to the cell trap. This is illustrated by Figure 7.
100431 As the seventh step, the power source electrically coupled to the electrode screws is turned on to create an electric field across the nanochannel array.
The electric field is created in the following fashion: the positive electrode is placed on the electrode screw that covers the holes that lead to the sample wells of the unused cell trap, while the negative electrode is placed on the electrode screw that covers the holes that lead to the sample wells of the used cell trap.
100441 These above mentioned steps provide quick and accurate isolation of the desired cells via the cell sorting microchannel from multiple cells supplied to the cell supply microchannel 1. Further, the steps mentioned above provide effective cell lysis and nucleic acid extraction from the desired cell and their movement into the nanochannel array.
[00451 According to the invention, the liquid containing the cells can flow through the cell supply microchannels quickly, driven by the supply mechanism. Thus, quick and accurate infusion of the cell suspension can be achieved.

100461 According to the invention, sorting of the desired cell from the T-junction to the cell sorting microchannel can be performed accurately with hydrostatic pressure manipulations after the supply mechanism stops. This is achieved by the following four steps: a first step of turning off the supply mechanism and stopping the cell suspension infusion, a second step of adjusting the valves at both cell supply microchannels so that the liquids in the cell supply microchannels are in direct contact with the atmospheric pressure and also subjected to hydrostatic pressure, a third step of varying the heights of the syringes to balance the hydrostatic pressure at the two cell supply microchannels to reduce net movement of liquids in the cell supply microchannels, and a fourth step of lowering the syringe connected to the cell exit microchannel and thus the hydrostatic pressure at the cell exit microchannel.
This induces a flow carrying the desired cell towards the cell trap. Thus, quick and selective single-cell isolation can be achieved.
100471 According to the invention, the cell trap consists of a barrier slit thinner than the average cell diameter, physical trapping the desired cell at the cell trap. The cell trap impedes the cell movement towards the cell exit microchannel. Thus, trapping of the desired cell from the cell sorting microchannel can be easily performed.
100481 According to the invention, undesired cells are prevented from flowing into the cell sorting microchannel so that only the desired cell can be surely sorted and to be extracted. This is achieved by: first, increasing the resistance at the cell trap that is created by the trapped cell of interest which blocks part of the cell trap, effectively impeding fluid flow from the cell supply microchannels to the cell trap, and second, increasing the vertical displacement of the syringe connected to the cell exit microchannel to increase the hydrostatic pressure at the cell exit microchannel, further impeding fluid flow from the cell supply microchannels to the cell trap, and third, pumping out the remaining cells in the cell supply microchannels with the supply mechanism.
100491 According to the invention, the desired cell can be treated with chemical lysis buffer and mechanical pressure for effective breaking of cell membrane and nuclear membrane. This is achieved by the following four steps: a first step of adjusting the valves that connect to the cell exit microchannel to impede fluid flow from the cell exit microchannel to the atmosphere or to the syringe connected to the cell exit microchannel, a second step of pumping chemical lysis buffer from cell supply microchannel 2, while adjusting the valve connected to the cell supply microchannel 1 to allow only fluid flow from the cell supply microchannel 1 to the atmosphere, replacing the existing cell samples in the cell supply channels with the lysis buffer, and a third step of adjusting the valves connected to the cell exit microchannel to allow only fluid flow from the cell exit microchannel to the atmosphere, and a fourth step of adjusting the valves connected to cell supply channel 1 to impede fluid flow from cell supply channel 1 to the atmosphere or to the syringe connected to cell supply channel 1, and using the supply mechanism to pump lysis buffer into cell supply microchannel 2, inducing a small flow across the barrier. This applies a mechanical shearing force on the cell membrane and the nuclear membrane at the cell trap. Thus, cell lysis can be achieved effectively and efficiently.
100501 According to the invention, the nucleic acids extracted from the desired cell can be directed into the barrier slit and trapped and sorted in the barrier slit. The desired nucleic acids are directed into the barrier slit by using the supply mechanism to induce a small flow from the cell supply microchannels to the cell trap.
The post array structures in the barrier slit effectively untangles and unwraps nucleic acid molecules, while trapping them in the barrier slit. The nucleic acid trapping structures in the barrier slit are not limited to post arrays. Other features such as nanopits can entropically trap nucleic acid molecules in the barrier slit. But the presented invention only includes post array structures.
100511 According to the invention, the nucleic acids extracted from the desired cell can be further sorted and separated into single molecules in the buffer zone.
This is achieved by post array structures in the buffer zone. The nucleic acid sorting structures in the buffer zone are not limited to post arrays. Other features such as a fan-shaped buffer zone can increase nucleic acid separation efficiency. But the presented invention only includes post array structures.
100521 The fused silica chips are fabricated on a four-inch fused silica wafer in clean room facilities. Each wafer accommodates nine chips; these are later diced using a wafer saw. The fabrication process is broken down into two stages; the nano-and the micro- features stage. The nanochannels are patterned using electron-beam lithography, while the microchannels are patterned using standard photolithography.
Nanofluidic features are dry-etched using reactive ion etching (RIE), which is highly anisotropic. Microfluidic features are wet etched using hydrofluoric acid (HF).
100531 A detailed fabrication workflow follows:
100541 As the first step, the wafer is spin coated with e-beam resist (ZEP520A) and the nano-features are defined by electron-beam lithography (JEOL).
100551 As the second step, the oxidized features are developed using PMMA
developer.
100561 As the third step, the nanochannels are etched into the silica using RIE with a CF4 :CHF3 gas combination at a flow rate ratio of 12:17 sccm. A 30s mild 02 plasma etch is performed before the etch to remove any debris remaining from the developed resist. We noticed that the RIE chamber needs to be cleaned using a strong 02 plasma etch for at least five minutes before etching; otherwise, the etched features could exhibit roughness due to chamber contamination.
100571 As the fourth step, the remaining resist is removed by sonicating in a hot bath of resist stripper (1165) at 65 C. To ensure the wafer is entirely clean, it is dropped in a piranha solution of sulfuric acid and hydrogen peroxide at ratio of 3:1.
This etches away any organic matter that might be remaining on the fused silica.
100581 As the fifth step, the wafer is first coated with HMDS primer to prevent the oxidation of its surface and allow the resist to better adhere. Then the wafer is spin coated with positive photoresist (S1813) and the micro-features are defined using contact ultraviolet photolithography using a chromium photomask.
100591 As the sixth step, the features are then developed in AZ-400K diluted in de-ionized water at a ratio of 1:4.
100601 As the seventh step, the features are wet etched using 35% hydrofluoric acid.
The time of hydrofluoric acid treatment is dependent on the desired etch depth.
Different embodiments of the invention require etch times from 20 minutes to 2 hours.
100611 As the eighth step, the resist is stripped as done in step 4.
100621 As the ninth step, loading holes are manually sandblasted through the device.
[00631 As the tenth step, lastly, the device is sealed using direct silica-silica bonding (RCAI and RCA2) to a 150 p.m thick silica cover slip (Valley Design). The chips are then treated using a bonding furnace, which bakes them to a 1000 C, to convert the hydrogen bonds to a more permanent covalent silicon-oxide bond.

Fig. 1 is a schematic top view of the fused silica chip.
Fig. 2 is an enlarged schematic view of the dotted area in Fig. 1 Fig. 3-7 are schematic views showing the device used for the method for cell sorting according to the present invention.
Fig. 3 shows the first step of the method for sorting single cells according to the present invention.
Fig. 4 shows the third step of the method for sorting single cells according to the present invention.
Fig. 5 shows the fourth step of the method for sorting single cells according to the present invention.
Fig. 6 shows the fifth step of the method for sorting single cells according to the present invention.
Fig. 7 shows the sixth step of the method for sorting single cells according to the present invention.
Fig. 8 is a schematic view showing the device used for the method for cell lysis according to the present invention.
Fig. 9 is a schematic view of the buffer zone and the nanochannel arrays Fig. 10 is a Scanning Electron Microscope photo of the nanochannel arrays and post arrays.
Fig. 11 is a photo view of the experiment setup.
Explanation on the Numerals 1 Cell Supply Microchannel 2 Cell Supply Microchannel 3 Cell Exit Microchannel 4 Cell Sorting Microchannel Sample Well 6 T-Junction 7 Cell Trap 8 Barrier Slit 9 Buffer Zone Nanochannel Arrays 11 Post Arrays 12 Chuck 13 Electronic Stage 14 Syringe (Supply Mechanism) 15 Valve 16 Tub ings 17 Inverted Fluorescence Microscope