Droplet array chip for cell-based assay and methods of manufacture

Abstract

A droplet array integrated circuit (IC) for biological cell analysis can include a semiconductor IC substrate with a hydrophobic region disposed on a first surface thereof. An array of hydrophilic regions can be positioned on the first surface, with individual hydrophilic regions bordered by the hydrophobic region. The IC can include a plurality of biological cell-adhering elements, with individual elements disposed atop corresponding hydrophilic regions of the array. The biological cell-adhering elements can be selective for adhering to specified cell types, sizes, shapes, or morphologies, enabling precise control over cell capture and positioning.

Description

Docket No. 3867.C75WO1DROPLET ARRAY CHIP FOR CELL-BASED ASSAY AND METHODS OF MANUFACTURECLAIM OF PRIORITY

[0001] This application claims priority to and the benefit of U.S. Provisional Application Serial No. 63 / 696,712, filed September 19, 2024, which is hereby incorporated herein by reference, and the benefit of priority of which is claimed herein.BACKGROUND

[0002] Droplet microfluidics provide an approach for conducting biological assays with enhanced precision and control compared to certain well plate-based systems. Certain approaches to assaying biological specimens involve well plates with standardized layouts and pitch of individual fluidic volumes, such as high throughput screening methods involving well plates having about 96, 384, or 1536 wells per plate.BRIEF DESCRIPTION OF THE DRAWINGS

[0003] In the drawings, which are not necessarily drawn to scale, like numerals can describe similar components in different views. Like numerals having different letter suffixes can represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed in the present document.

[0004] FIG. 1 A is a top view of a droplet array chip for holding a single cell in hydrophilic regions.

[0005] FIG. IB is a top view of a droplet array chip for holding a plurality of cells in hydrophilic regions.

[0006] FIG. 1C is a top view of a droplet array chip for holding a plurality of cells in hydrophilic regions.

[0007] FIG. 2A depicts an approach to forming a droplet array integrated circuit (IC).

[0008] FIG. 2B depicts an approach to forming a droplet array integrated circuit (IC)

[0009] FIG. 3 depicts selective loading of biological cells into individual droplet on droplet array integrated circuit (IC).

[0010] FIG. 4 is a flowchart showing a process 400 for selectively loading biological cells into individual droplet on droplet array integrated circuit (IC).Docket No. 3867.C75WO1

[0011] FIG. 5 is a block diagram of a machine.DETAILED DESCRIPTION

[0012] Droplet array integrated circuits (ICs) can be used to receive biological specimens thereon, such as via an array of droplets formed on a face of the IC. Loading individual cells on a droplet array integrated circuit (IC) with a desired specificity can be challenging. For example, it can be difficult to achieve a desired cell loading percentage, as well as challenging to load a droplet array integrated circuit (IC) that without being substantially limited by a Poisson distribution of cells on the surface of the IC. Certain other droplet-based cell assays can utilize random loading of cells where the cell number cannot be controlled per droplet, such as inherently limited by a Poisson distribution process. Such a Poisson distribution of cells across a surface of the IC can inhibit certain approaches to continuous monitoring or controlling the position of cells within droplets on the surface of the IC. Additionally, certain other single cell-based assay loading approaches can result in numerous empty droplets that must be filtered out, presenting additional processing steps and resulting in substantial waste of reagents and droplet volumes. The inability to precisely control the number of cells per droplet represents a fundamental limitation can ultimately reduce assay efficiency and increase operational costs.

[0013] The present inventors have recognized the benefits of a droplet array integrated circuit (IC) that provides precise control over cell number and position within individual droplets. The droplet array IC can include a semiconductor IC substrate with a hydrophobic region disposed on a first surface of the IC substrate. An array of hydrophilic regions can be positioned on the first surface, with individual hydrophilic regions bordered by the hydrophobic region. A plurality of biological cell-adhering elements can be disposed atop corresponding hydrophilic regions of the array. In an example, these biological cell-adhering elements can be selective for adhering to specified cell types, sizes, shapes, or morphologies, enabling precise control over which cells are captured and positioned within each droplet.

[0014] FIG. 1A, FIG. IB, and FIG. IC each provide a top view of a respective droplet array chip or integrated circuit (IC) 102 for holding a single cell in hydrophilic regions 110. In an example, an IC 102 can include a hydrophobic region 108 and a plurality of hydrophilic regions 110 disposed within and separated from one another by the 108. The hydrophilic regions 110 can include an antifouling or cell repelling material, e.g., polyethylene glycol (PEG) or silane-functionalized PEG derivatives. For example, PEG can provide as an antifouling layer that prevents attachment of proteins, cells, DNA, and other biological molecules, such that during loading of cells onto the IC 102, cells have nowhere to attach except withinDocket No. 3867.C75WO1 specified regions of the IC specifically designed for cell adhesion. For example, biological cell-adhering cell adhering elements 112 can include at least one of extracellular matrix (ECM) proteins, polyelectrolytes, antibody proteins, or cell-adhesion peptides. Specific ECM proteins can include fibronectin or laminin. When ECM proteins are provided or arranged in regions without PEG molecules, cells will selectively attach to these designated areas.

[0015] In an example, the hydrophobic region 108 can include fluoroalkylsilane, though alternative hydrophobic coatings such as parylene, FluoroPei, or Teflon can be included or used. Individual hydrophilic regions 110 of the array of hydrophilic regions 110 can have a greatest diameter within a range of about 100 micrometers to about 200 micrometers, while individual cell-adhering cell adhering elements 112 can span a greatest diameter within a range of about 5 micrometers to about 15 micrometers. Such as size and shape of the hydrophilic region 110 and the cell adhering element 112 can help facilitate selectivity and control over the number of cells captured, as the cell-adhering area can be designed to be just large enough to capture the desired number of cells, such as promoting predictable and desired cell occupancy per droplet array. For example, as shown by the various examples in FIG. 1 A, FIG. IB, and FIG. IC, an amount of cell adhering elements 112 positioned or disposed within an a droplet at an individual hydrophilic region 110 can substantially determine an amount of cells that will adhere to that IC 102, e.g., about one cell per droplet in FIG. 1A, about four cells per droplet in FIG. IB, and about two cells per droplet in FIG IC. As shown in FIG. IC, in an example an individual hydrophilic region 110 can include a first cell adhering element 112 and a second, cell adhering element 114. Here, the first cell adhering element 112 can be selective for a different cell type (e.g., size, morphology, etc.) than that of the second cell adhering element 114. Thus, an individual hydrophilic region 110 can be selective for two different types of cells concurrently, while still controlling placement and amount of each of the two different types of cells during loading.

[0016] FIG. 2A and FIG. 2B each depict respective processes 200A and 200B for forming a droplet array integrated circuit (IC).

[0017] Referring to FIG. 1A, at 210, the process 200A can include coating an IC substrate 228 (e.g., silicon) with a photoresist 232. At 212, portions of the photoresist 232 can be displaced from the IC substrate, e.g., via manual removal, irradiation, etching, etc., such as to define cavities 226 that expose the underlying IC substrate. At 214, a hydrophobic material 230 (e.g., fluoroalkylsilane) can be transferred (e.g., via photolithography) such as to fill the cavities 226 as well as bind to and overlay atop the photoresist. At 216, the bound photoresist 232 and hydrophobic material 230 can be lifted off the substrate. At 218, a hydrophilic material 234 (e.g., PEG-silane) can fill where the bound photoresist 232 and hydrophobicDocket No. 3867.C75WO1 material 230 were removed (e.g., between remaining portions of the hydrophilic material 234, still embedded on the substrate. At 220, biological cell-adhering elements 236 (e.g., extracellular matrix proteins, polyelectrolytes, antibody proteins, cell-adhesion peptides, or a combination thereof) can be embedded into the hydrophilic material 234, e.g., via a polydimethylsiloxane (PDMS) stamp 238, establishing the IC 102.

[0018] Referring to FIG. IB, at 240, the process 200B can include coating an IC substrate 228 with a hydrophobic material 230 (e.g., fluoroalkylsilane). At 242, UV radiation (or other type of photolithographic technique) can be applied to remove at least a portion of the hydrophobic material 230, exposing cavities 226 in the hydrophobic material 230 and revealing the IC substrate 228. At 244, these cavities 226 can be filled with a hydrophilic material 234 (e.g., PEG-silane), establishing the IC 102. Here, due to the specificity in patterning provided by the UV radiation, an additional cell-adhering element need not necessarily be embedded on the IC 102, as a hydrophobic spot 246 remains after forming the droplet array IC 102.

[0019] FIG. 3 depicts selective loading of biological cells into individual droplet on droplet array integrated circuit (IC). Such selective loading of an IC 102 can involve flowing a first liquid solution 312 (e.g., an assay medium such as phosphate buffered saline (PBS), including suspended biological cells 310 over the droplet array IC, where the IC 102 includes the hydrophobic region and array of hydrophilic regions with biological cell-adhering elements disposed thereon. At least a portion of the first liquid solution 312 can be removed from the IC to form liquid droplets 308 atop the hydrophilic regions.

[0020] Suspended biological cells bind to respective biological cell-adhering elements disposed on the array of hydrophilic regions during an incubation period. As depicted in FIG. 3, due to only one cell-adhesive area being disposed at the center of each hydrophilic region, there is a high likelihood that only one cell will attach in configurations designed for singlecell occupancy. A second liquid solution 314 can be then flowed over the IC to rinse unbound biological cells, removing all free cells that have not adhered to the designated cell-adhering elements. Finally, at least a portion of the second liquid solution 314 is removed from the IC to reform liquid droplets atop the hydrophilic regions, wherein the reformed liquid droplets 316 contain biological cells bound to respective cell-adhering elements. This approach enables the creation of droplet arrays where only one cell is maintained per droplet, or a precisely controlled number of cells based on the design of the cell-adhering regions.

[0021] FIG. 4 is a flowchart showing a process 400 for selectively loading biological cells into individual droplet on droplet array integrated circuit (IC).Docket No. 3867.C75WO1

[0022] At 402, the process 400 can include flowing a first liquid solution including suspended biological cells over the droplet array IC, the droplet array IC including a first surface including a hydrophobic region and an array of hydrophilic regions bordered by hydrophobic region. Here, the array of hydrophilic regions each include a biological celladhering element disposed thereon.

[0023] At 404, the process 400 can include removing at least a portion of the first liquid solution from the IC form liquid droplets atop the hydrophilic regions.

[0024] At 406, the process 400 can include binding suspended biological cells to respective biological cell-adhering elements disposed on the array of hydrophilic regions.

[0025] At 408, the process 400 can include flowing a second liquid solution to rinse unbound biological cells from the IC.

[0026] At 410, the process 400 can include removing at least a portion of the second liquid solution from the IC to reform liquid droplets atop the hydrophilic regions. Here, the reformed liquid droplets contain biological cells bound to respective cell-adhering elements. In an example, the biological cell-adhering element can have a greatest diameter within a range of 5 micrometers (pm) and 15 pm and the individual cavity of hydrophobic material, filled by the hydrophilic material, can defines a region having a greatest diameter within a range of 100 pm and 200 pm.

[0027] FIG. 5 illustrates generally an example of a block diagram of a machine 501 upon which any one or more of the techniques (e.g., methodologies) discussed herein may perform in accordance with some examples. In alternative embodiments, the machine 501 may operate as a standalone device or may be connected (e.g., networked) to other machines. In a networked deployment, the machine 501 may operate in the capacity of a server machine, a client machine, or both in server-client network environments. In an example, the machine 501 may act as a peer machine in peer-to-peer (P2P) (or other distributed) network environment. The machine 501 may be a personal computer (PC), a tablet PC, a set -top box (STB), a personal digital assistant (PDA), a mobile telephone, a web appliance, a network router, switch or bridge, or any machine capable of executing instructions (sequential or otherwise) that specify actions to be taken by that machine. Further, while only a single machine is illustrated, the term “machine” shall also be taken to include any collection of machines that individually or jointly execute a set (or multiple sets) of instructions to perform any one or more of the methodologies discussed herein, such as cloud computing, software as a service (SaaS), other computer cluster configurations.Docket No. 3867.C75WO1

[0028] Examples, as described herein, may include, or may operate on, logic or a number of components, modules, or mechanisms. Modules are tangible entities (e.g., hardware) capable of performing specified operations when operating. A module includes hardware. In an example, the hardware may be specifically configured to carry out a specific operation (e.g., hardwired). In an example, the hardware may include configurable execution units (e.g., transistors, circuits, etc.) and a computer readable medium containing instructions, where the instructions configure the execution units to carry out a specific operation when in operation. The configuring may occur under the direction of the executions units or a loading mechanism. Accordingly, the execution units are communicatively coupled to the computer readable medium when the device is operating. In this example, the execution units may be a member of more than one module. For example, under operation, the execution units may be configured by a first set of instructions to implement a first module at one point in time and reconfigured by a second set of instructions to implement a second module.

[0029] Machine (e.g., computer system) 501 may include a hardware processor 502 (e.g., a central processing unit (CPU), a graphics processing unit (GPU), a hardware processor core, or any combination thereof), a main memory 503 and a static memory 504, some or all of which may communicate with each other via an interlink (e.g., bus) 505. The machine 501 may further include a display unit 506, an alphanumeric input device 507 (e.g., a keyboard), and a user interface (UI) navigation device 508 (e.g., a mouse). In an example, the display unit 506, alphanumeric input device 507 and ui navigation device 508 may be a touch screen display. The machine 501 may additionally include a storage device (e.g., drive unit) 509, a signal generation device 510 (e.g., a speaker), a network interface device 511, and one or more sensors 512, such as a global positioning system (GPS) sensor, compass, accelerometer, or other sensor. The machine 501 may include an output controller 516, such as a serial (e.g., universal serial bus (USB), parallel, or other wired or wireless (e.g., infrared (IR), near field communication (NFC), etc.) connection to communicate or control one or more peripheral devices (e.g., a printer, card reader, etc.).

[0030] The storage device 509 may include a machine readable medium 513 that is non- transitory on which is stored one or more sets of data structures or instructions 514 (e.g., software) embodying or utilized by any one or more of the techniques or functions described herein. The instructions 514 may also reside, completely or at least partially, within the main memory 503, within static memory 504, or within the hardware processor 502 during execution thereof by the machine 501. In an example, one or any combination of the hardware processor 502, the main memory 503, the static memory 504, or the storage device 509 may constitute machine readable media.Docket No. 3867.C75WO1

[0031] While the machine readable medium 513 is illustrated as a single medium, the term “machine readable medium” may include a single medium or multiple media (e.g., a centralized or distributed database, or associated caches and servers) configured to store the one or more instructions 514.

[0032] The term “machine readable medium” may include any medium that is capable of storing, encoding, or carrying instructions for execution by the machine 501 and that cause the machine 501 to perform any one or more of the techniques of the present disclosure, or that is capable of storing, encoding or carrying data structures used by or associated with such instructions. Non-limiting machine-readable medium examples may include solid-state memories, and optical and magnetic media. Specific examples of machine-readable media may include: non-volatile memory, such as semiconductor memory devices (e.g., Electrically Programmable Read-Only Memory (EPROM), Electrically Erasable Programmable Read- Only Memory (EEPROM)) and flash memory devices; magnetic disks, such as internal hard disks and removable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks.

[0033] The instructions 514 may further be transmitted or received over a communications network 515 using a transmission medium via the network interface device 511 utilizing any one of a number of transfer protocols (e.g., frame relay, internet protocol (IP), transmission control protocol (TCP), user datagram protocol (UDP), hypertext transfer protocol (HTTP), etc.). Example communication networks may include a local area network (LAN), a wide area network (WAN), a packet data network (e.g., the Internet), mobile telephone networks (e.g., cellular networks), Plain Old Telephone (POTS) networks, and wireless data networks (e.g., Institute of Electrical and Electronics Engineers (IEEE) 802.11 family of standards known as Wi-Fi®, IEEE 802.16 family of standards known as WiMax®), IEEE 802.15.4 family of standards, peer-to-peer (P2P) networks, among others. In an example, the network interface device 511 may include one or more physical jacks (e.g., Ethernet, coaxial, or phone jacks) or one or more antennas to connect to the communications network 515. In an example, the network interface device 511 may include a plurality of antennas to wirelessly communicate using at least one of single-input multiple-output (SIMO), multiple-input multiple-output (MIMO), or multiple-input single-output (MISO) techniques. The term “transmission medium” shall be taken to include any intangible medium that is capable of storing, encoding or carrying instructions for execution by the machine 501, and includes digital or analog communications signals or other intangible medium to facilitate communication of such software.

Claims

Docket No. 3867.C75WO1CLAIMSWhat is claimed is:

1. A droplet array integrated circuit (IC) comprising: a semiconductor IC substrate; a hydrophobic region disposed on the IC substrate, the hydrophobic region disposed on a first surface of the IC substrate; an array of hydrophilic regions on the first surface of the IC substrate, individual ones of the hydrophilic regions bordered by the hydrophobic region on the first surface; a plurality of biological cell-adhering elements, individual ones disposed atop a corresponding hydrophilic region of the array of hydrophilic regions; and wherein the plurality of biological cell-adhering elements are selective for adhering to a specified cell type, size, shape, or morphology.

2. The IC of claim 1, wherein an individual hydrophilic region of the array of hydrophilic regions includes polyethylene glycol (PEG) or a silane-functionalized PEG derivative.

3. The IC of any of claims 1-2, wherein an individual cell-adhering element of the plurality of biological cell-adhering elements includes at least one of an extracellular matrix (ECM) protein, a polyelectrolytes, an antibody protein, or a cell-adhesion peptide.

4. The IC of any of claims 1-3, wherein an individual cell-adhering element of the plurality of biological cell-adhering elements includes at least one of an extracellular matrix (ECM) protein including at least one of fibronectin or laminin.

5. The IC of any of claims 1-4, wherein the hydrophobic region includes fluoroalkylsilane.

6. The IC of any of claims 1-5, wherein an individual hydrophilic region of the array of hydrophilic regions has a greatest diameter within a range of 100 micrometers (pm) and 200 pm.

7. The IC of any of claims 1-6, wherein an individual cell-adhering element of the plurality of biological cell-adhering elements has a greatest diameter within a range of 5 micrometers (pm) and 15 pm.

8. A method for selectively loading biological cells into individual droplet on droplet array integrated circuit (IC), the method comprising:Docket No. 3867.C75WO1 flowing a first liquid solution including suspended biological cells over the droplet array IC, the droplet array IC including a first surface including a hydrophobic region and an array of hydrophilic regions bordered by hydrophobic region, wherein the array of hydrophilic regions each include a biological cell-adhering element disposed thereon; removing at least a portion of the first liquid solution from the IC form liquid droplets atop the hydrophilic regions; binding suspended biological cells to respective biological cell-adhering elements disposed on the array of hydrophilic regions; flowing a second liquid solution to rinse unbound biological cells from the IC; and removing at least a portion of the second liquid solution from the IC to reform liquid droplets atop the hydrophilic regions, wherein the reformed liquid droplets contain biological cells bound to respective cell-adhering elements.

9. The method of claim 8, wherein an individual hydrophilic region of the array of hydrophilic regions includes polyethylene glycol (PEG) or a silane-functionalized PEG derivative.

10. The method of any of claims 8-9, wherein the biological cell-adhering element includes at least one of an extracellular matrix (ECM) protein, a polyelectrolytes, an antibody protein, or a cell-adhesion peptide.

11. The method of any of claims 8-10, wherein the biological cell-adhering element includes at least one of an extracellular matrix (ECM) protein including at least one of fibronectin or laminin.

12. The method of any of claims 8-11, wherein the hydrophobic region includes fluoroalkylsilane.

13. The method of any of claims 8-12, wherein the first and second liquid solutions each include an assay medium.

14. The method of any of claims 8-13, wherein the assay medium includes phosphate buffered saline (PBS).

15. A method for manufacturing a droplet array integrated circuit (IC), the method comprising: coating an IC substrate with a hydrophobic material;Docket No. 3867.C75WO1 displacing portions of the hydrophobic material from the IC substrate to define cavities in the hydrophobic material, the cavities exposing the IC substrate; transferring, via photolithography, a hydrophilic material to fill the cavities of the hydrophobic material; and embedding, via a polydimethylsiloxane (PDMS) stamp, to embed a biological celladhering element to the hydrophilic material.

16. The method of claim 15, wherein the hydrophilic material includes polyethylene glycol (PEG) or a silane-functionalized PEG derivative.

17. The method of any of claims 15-16, wherein the biological cell-adhering element includes at least one of an extracellular matrix (ECM) protein, a polyelectrolytes, an antibody protein, or a cell-adhesion peptide.

18. The method of claim 17, wherein the biological cell-adhering element includes at least one of an extracellular matrix (ECM) protein including at least one of fibronectin or laminin.

19. The method of any of claims 15-18, wherein the hydrophobic material includes fluoroalkylsilane.

20. The method of any of claims 15-19, wherein an individual cavity of hydrophobic material, filled by the hydrophilic material, defines a region having a greatest diameter within a range of 100 micrometers (pm) and 200 pm.

21. The method of any of claims 15-20, wherein the biological cell-adhering element has a greatest diameter within a range of 5 micrometers (pm) and 15 pm.

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