Methods of making and using chiplets

Abstract

Described herein are methods and devices for creating and using chiplets loaded with different reagents embedded in a gel carrier material. Microfluidic devices are used to create chiplets. The reagents embedded in the gel carrier materials are drawn through the channels of the microfluidic device and then secured in place within the microfluidic device. One or more chiplets are separated from the region of interest portion of the microfluidic device after the reagents are fixed in place, typically by lowering the temperature. These chiplets may then be packaged and sold to end users for a variety of applications where microfluidic devices are traditionally required. The end user may then secure one or more chiplets to the sample without performing microfluidic flow operations.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 63 / 427,050, filed on Nov. 21, 2022, entitled "CHIPLET PRODUCTION AND METHODS OF USE", and U.S. Provisional Patent Application Ser. No. 63 / 427,052, filed on Nov. 21, 2022, entitled "CHIPLET PRODUCTION AND METHODS OF USE". The entire content of each of these applications is incorporated herein by reference. Background of the Invention Field of the Invention

[0003] The present disclosure generally relates to the preparation and use of chiplets produced using microfluidic devices. More specifically, the channels of the microfluidic device can be loaded with a hydrogel that is impregnated with an active ingredient intended to be applied to a target substrate. This transforms the microfluidic device into a substrate with demarcated reagents separated by the channel boundaries. These chiplets can improve spatial resolution and precision compared to flowing reagents over the microfluidic device and the target substrate. Additionally, the chiplets can be cut or segmented to form or produce multiple chiplets from a single chiplet.

[0004] Related Art

[0005] Microfluidics can be used to perform many different applications. One particular application of microfluidics, commonly referred to as spatially multiplexed omics sequencing of deterministic barcodes in tissue (DBiT - seq), requires placing a thin array of empty channels made of silicone on top of the tissue, pressing them down in a uniform and precise manner to create a seal against the tissue surface without collapsing the channel walls, and then pumping reagents through the channels to wash the reagents in precise geometric regions on the tissue section. In practice, this delivers sufficient active ingredients (e.g., oligonucleotides, enzymes, fluorophores, collectively referred to hereinafter as "reagents") to perform a series of spatially resolved omics techniques, such as spatial transcriptomics for messenger RNA (mRNA) localization, spatial proteomics for proteome localization, spatial epigenomics for chromatin accessibility / histone modification localization, and various combinations thereof. Summary of the Invention

[0006] One embodiment can provide a microfluidic chip, the microfluidic channels of which are filled with a set of hydrogel strips with different loads. Each strip can accommodate one of a series of reagents, for example, an oligonucleotide with a specific sequence indicating the number of channels, or the reagent concentration in a titration screening. After peeling from the flow substrate, such a loaded microfluidic chip (referred to herein as a "small chip") can then be used as a stamp to deliver the active ingredient to the target substrate in a spatially defined manner (in a preferred embodiment, the substrate is a tissue section mounted on a coated glass slide).

[0007] In another embodiment, the small chip can be released from the inactive area of the microfluidic device (e.g., by cutting the small chip). This allows the active area of the small chip to be significantly increased. In addition, this largely eliminates the size limitations on the periphery of the microfluidic chip. More importantly, this allows the small chip to be divided into multiple small chips (similar to semiconductors), where the time and cost consumed by each small chip can be further reduced.

[0008] A method for manufacturing a small chip is described, which includes the following steps: directly fixing a flow substrate to a microfluidic chip including a plurality of channels; simultaneously flowing a first reagent embedded in a first gel carrier material through a first channel among the plurality of channels and flowing a second reagent embedded in a second gel carrier material through a second channel among the plurality of channels; adjusting one or more material properties of the first gel carrier material and the second gel carrier material provided in the plurality of channels to limit the further movement of the first reagent and the second reagent within the microfluidic chip; and optionally cutting the microfluidic chip into multiple pieces to produce at least a first small chip, where the first small chip includes a first part of the first channel and a second part of the second channel, and the first part and the second part of the first channel and the second channel included in the first small chip extend from a first end of the first small chip to a second end of the first small chip.

[0009] In one embodiment, the microfluidic chip can be customized to increase its affinity for the hydrogel (e.g., increasing the hydrophilicity of the microfluidic channels).

[0010] In another embodiment, the gel can be customized to increase its affinity for the microfluidic device (e.g., increasing its hydrophobicity).

[0011] Describes a method of using a microlchip, and the method includes the following steps: applying the microlchip to a tissue sample, where the microlchip includes a plurality of parallel channels extending from a first end of the microlchip to a second end opposite the first end, the plurality of channels including a first channel and a second channel, the first channel being filled with a first reagent embedded in a first gel carrier material, and the second channel being filled with a second reagent embedded in a second gel carrier material; clamping the microlchip to the tissue sample; raising the temperature of the microlchip to allow the first reagent and the second reagent to flow onto the tissue sample and interact with the tissue sample; and removing the microlchip from the tissue sample after a predetermined incubation time.

[0012] Discloses a microlchip, and the microlchip includes: a first substrate defining a plurality of channels, where the plurality of channels extend from a first end of the substrate to a second end opposite the first end; a first reagent embedded in a gel carrier material filling the first channel among the plurality of channels; a second reagent embedded in the gel carrier material filling the second channel among the plurality of channels; and a second substrate covering the plurality of channels. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] This patent or application document contains at least one color drawing. A copy of the patent application disclosure with color drawings will be provided by the patent office upon request by the requester and payment of the necessary fees.

[0014] Figure 1 Shows successful flow in a 50-channel 25-micron microfluidic chip;

[0015] Figure 2 Is a schematic diagram of a flow-based process (left) and a microlchip-based process (right) for delivering spatial barcodes to tissue sections.

[0016] Figures 3A to 3C Shows images of three different 50-channel 25-um microlchips captured for quality inspection after peeling.

[0017] Figure 4A Shows an exploded view of the chip cassette.

[0018] Figure 4B Shows being set in Figure 4A Perspective view of the microlchip in the chip cassette depicted.

[0019] Figure 5 Shows the active and inactive regions of the DBiT-seq microfluidic device.

[0020] Figures 6A to 6B Shows delivery of fluorescently labeled oligonucleotides to two different fresh frozen mouse embryo sections.

[0021] Figure 7 Four electropherograms showing head-to-head comparisons of multiple spatial ATAC-seq runs.

[0022] Figures 8A to 8D TIXEL maps of fragment counts showing head-to-head comparisons of two flow-based spatial ATAC-seq runs and two matched chip-based runs.

[0023] Figure 9A A sequencing statistics data table showing comparisons between matched flow-based runs and chip-based runs.

[0024] Figures 9B to 9E More results of chip-based DBiT runs are shown.

[0025] Figure 10 A microfluidic chip with a "superchip" design characterized by 288 individual inlets is shown.

[0026] Figure 11 An image of a microfluidic chip with a master chip design configured to fabricate multiple chips is shown.

[0027] Figure 12 A common way of mounting multiple tissue sections on a single slide is shown.

[0028] Figure 13 A microfluidic chip with a serpentine design is shown.

[0029] Figure 14 Shows Figure 13 An intensity map of the area within the dashed square in

[0030] Figure 15 Another microfluidic chip with a "master chip" design is shown.

[0031] Figures 16A to 16B A configuration is shown in which a tissue sample is located between Chip A and Chip B.

[0032] Figures 17A to 17B A method for depositing barcoded gels on a solid substrate is shown.

[0033] Figure 18 The application of electrophoresis for delivering an active ingredient from a gel carrier into a target substrate is shown.

[0034] Figures 19A to 19C Relative cycle time data for DBiT operations are shown.

[0035] Figures 20A to 20BShows multiple different electrophoresis maps of chiplet-based DBiT runs.

[0036] Figures 21A to 21C Shows an exemplary sequencing dataset generated using chiplet-based processing.

[0037] Figures 22A to 22G Shows a comparison of chiplet-based runs and standard DBiT-ATAC-seq runs performed on tissue sections from the same sample block.

[0038] Figure 23 Depicts side-by-side comparison data of ATAC-DBiT-seq data collected from tissue sections from the same tissue blocks (mouse hippocampus and mouse cerebellum).

[0039] Figure 24 Displays spatially resolved transcriptome and ATAC data co-profiled on the same tissue section using chiplets.

[0040] Figure 25 Shows an image of the chip surface with fluorescently impregnated barcode reagents printed on a cell matrix on a glass slide. Detailed Description

[0041] The following description is presented to enable a person skilled in the art to make and use embodiments, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the disclosure. Thus, the disclosed system is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0042] Introduction

[0043] When a microfluidic chip is engaged on a substrate (such as a tissue section) with a clamping pressure typically in the range of 0 to 50 pounds per square inch (PSI), each channel of the microfluidic chip is typically formed or defined by three walls of PDMS or silicone and another wall of tissue / glass. The cross-section of the resulting channels ranges from 10×10 µm to 50×50 µm. These channels are typically arranged in parallel, and ideally, a liquid carrying one or more reagents is aspirated through each channel of the microfluidic chip without flow irregularities.

[0044] Figure 1Shows successful flow in a 50-channel 25×25 µm chip (meaning there are 50 channels, each with a 25×25 micron cross-section). The reagent flowing here is a 30-nt ssDNA oligonucleotide conjugated to one of two fluorophores (red (Cy3) and green (FAM)). The chip is clamped onto a mouse embryo tissue section mounted on a poly-L-lysine (PLL)-coated glass slide.

[0045] While Figure 1 the examples in show that it is possible to have unobstructed, leak-free flow on a range of tissues and tissue preparation methods using a 25 µm microfluidic chip, in many cases, due to uncontrollable differences during tissue section preparation and fixation, it is difficult for even the most skilled operator to achieve "perfect flow" (i.e., all channels are flowing and there is no leakage between channels). In production runs (i.e., spatial ATAC-seq), using existing technology methods and equipment, the proportion of channels that show good flow (neither leakage nor blockage) for a 25 µm microfluidic chip is close to 97%.

[0046] Now consider a 10 µm microfluidic chip. It provides the much-needed quasi-cellular-level spatial resolution in the industry but at the cost of flow performance. However, at this high resolution, approximately half of the flow tests and full DBiT with high-quality 10 µm chips suffer significant flow irregularities, which are defined as >5% of the channels having leakage or blockage.

[0047] One solution to this performance problem is to stop flowing on the tissue surface. While current DBiT-seq technology does flush reagents over the tissue, this is not a core feature of the technology. Its core feature is to deliver reagents to the substrate in a geometrically precise manner and then use combinations of these geometries to generate uniquely labeled regions. This principle holds regardless of the liquid deposition method - whether through a microfluidic chip, a microbrush, or other techniques that can achieve precise reagent positioning.

[0048] The core function of a DBiT-seq microfluidic chip is to precisely print thin lines of active reagents on a rough substrate. We determined that without attempting to print on a rough substrate, the DBiT microfluidic platform is fully capable of preparing lines as thin as 10 microns or even 5 microns or finer - these lines cross-printed can form regions as small as 10×10 µm, 5×5 µm or even smaller. Therefore, we determined that by filling the microchannels of a DBiT-seq chip with a suitably barcoded liquid hydrogel on a smooth substrate, one or more parts of a pre-loaded microfluidic chip can be optionally extracted, including those parts of the microchannels that would normally contact the tissue sample to produce a stamp or what we will refer to as a microchip in this specification.

[0049] This discovery led to the development of the embodiments described herein. These embodiments provide biologists with a user-friendly way to apply reagents to tissue sections with cellular-level precision and a very low risk of inaccurate barcoding. Different from the standard DBiT-seq technology, biologists do not need to perform microfluidic operations that usually require skilled operators to complete under ideal conditions. Nor is it necessary to perform liquid rinsing on the tissue surface; instead, the filling process of the small chip is completed under ideal conditions on a smooth substrate. Subsequently, the small chip is removed from the smooth substrate, inspected for quality, and then imprinted onto the tissue. Instead of asking biologists to manually control the flow process of each set of barcode matrices, we have constructed a truly spatial omics barcode printing system.

[0050] Flow in Factory / Imprint in Laboratory

[0051] The described embodiments divide the process of printing reagents onto tissue into the following steps: (1) filling a small chip with a gel impregnated with the reagent; (2) peeling the small chip from the flow substrate, inspecting the quality, and then storing and shipping it to the end user; and (3) the end user unpacks the small chip and docks the small chip with the substrate selected by the end user. This process is schematically depicted in Figure 2 .

[0052] Figure 2 is a schematic diagram of the flow-based process (left) and the small-chip-based process (right) for delivering spatial barcodes to tissue sections. In the small-chip-based process, a microfluidic chip is filled with barcoded gel in a small-chip factory. After flowing and removing the small chip from the smooth flow substrate, the quality of the small chip is visually inspected. Small chips that do not meet the quality threshold are discarded. Although this practice will reduce the manufacturing yield, it can protect precious tissue samples and save the time consumed by biologists / laboratories due to imperfect barcode arrays. Small chips that pass the quality inspection are packaged and sent to the end user, who uses these small chips to imprint the barcode array onto the substrate they have selected.

[0053] This modular design stands in stark contrast to traditional flow-based DBiT-seq methods; the latter require end-users to manipulate liquid flow within the chip themselves under non-ideal conditions, often resulting in flow irregularities that reduce the quality and / or quantity of data obtained from a particular sample. Each flow operation (two for each sample) puts at risk all the resources required to successfully analyze that sample. Poor flow results waste tissue samples, reagents, laboratory man-hours, biologists' time, and even the entire experimental design; this is particularly severe for precious and irreplaceable tissue samples. Even disregarding the cost of the tissue samples themselves, the combined losses per single test can amount to over a thousand dollars. When dealing with precious, irreplaceable tissue sections, these cost losses can be incalculable, and thus every effort must be made to ensure high-quality biological data is obtained.

[0054] The described embodiments create a small chip in a flow-friendly environment using the following exemplary steps: (1) Dock a fresh, clean microfluidic chip with a flow substrate. (2) Preheat the chip / glass slide assembly on a hotplate to ≥37 °C. (3) Meanwhile, heat a 96-well PCR plate, with 50 wells filled with barcode-gelatin mixture. (4) Pipette the appropriate barcoded gel into each well. Then, aspirate the barcoded hydrogel into the respective channels of the microfluidic chip by vacuum negative pressure. The gel continuously flows through the channels for a predetermined amount of time (usually between 2 and 10 minutes) at a predetermined vacuum pressure (usually between -40 and -80 inches of water column) until the barcoded gel uniformly fills the channels within the region of interest (i.e., the part of the microfluidic chip that will usually contact the tissue sample). (5) Then allow the gel to cool and solidify within the channels (either by passive cooling or by active cooling using ice, dry ice, placing in a refrigerator, docking with any cold substrate, etc.), and this process can be carried out before or after turning off the vacuum. (6) Then peel the flow substrate from the filled small chip and visually inspect the small chip (in an epifluorescence microscope) to check for any defects in the active area. (7) Small chips without defects are stored and kept for later use. Defective small chips are discarded.

[0055] It should be noted that in some embodiments, the flow substrate can remain against the microfluidic chip and be removed only when the customer is ready to utilize the small chip. In this case, the small chip can be formed by simultaneously cutting the microfluidic chip and the flow substrate. Alternatively, the party responsible for creating the small chip can peel the flow substrate from the small chip and then cover the channels of the small chip with another protective layer to prevent contamination of the reagents contained within the channels.

[0056] In some embodiments, the microfluidic chip (or "chiplet") is made of a hydrophilic material or treated to make the microfluidic surface hydrophilic, thereby reducing flow resistance and enhancing the binding between the gel and the chip (or chiplet).

[0057] Clogging and leakage phenomena during the chiplet filling process are typically significantly reduced compared to conventional DBiT techniques that flow over tissue. This is because the flow substrate is known at this stage (e.g., blank glass). This performance improvement has been observed in the case of 10um, 25um, and 50um resolution chips, and in one test in the case of a 5um resolution chip. This improvement is achieved because there is no tissue or other rough substrate impeding the flow, and the channels can conduct the liquid-phase gel almost perfectly from one end of the chip to the other. For a glass slide without tissue as the substrate, we can clean it quickly and effectively to ensure that no residual debris interferes with the flow.

[0058] If any flow failures occur, it will reduce the manufacturing yield, resulting in a negative economic impact. Due to air bubbles or defects in the microfluidic chip, such errors may still occur (of course, the incidence rate is extremely low because we carefully pipette and manually screen for defects in the chip). In any case, effective quality control steps can isolate the impact of flow failures on downstream detection. If the approximate production yield is known, simply multiply the production volume by the reciprocal of the expected yield (i.e., 1 / expected yield) for overproduction to correct for the losses.

[0059] It should be noted that before docking the chiplet with the tissue, it must first be removed from the original flow substrate on which it was carried. This step introduces another manufacturing failure mode, which is typically characterized by the gel strips adhering to the flow substrate as well as or even better than to the PDMS walls of the microchannels that form the chiplet. This failure mode can be observed by examining the peeled chiplet under a microscope. The "noodling" phenomenon (i.e., the gel strips are missing or scattered disorderly in the channels after being pulled out of the channels) will cause the chiplet to fail and must be scrapped. Examples of noodling can be seen in Figures 3A to 3C .

[0060] Figures 3A to 3C Images of three different 50-channel 25um chiplets captured for quality inspection after peeling are shown. The gel is impregnated with an active reagent and a red (ROX) or green (FITC) inert fluorescent dye to indicate the presence or absence of the gel. The images are generated by imaging the chip after peeling from the substrate using a two-color epi-fluorescence microscope. Specifically, Figure 3A shows a failed peel where most of the gel strips have been torn out of the microchannels. Figure 3BShows a failed peel where the gel strip towards the left of the region of interest is torn out of the microchannel. The small chip is discarded before docking with the tissue. Figure 3C Shows a successful peel where all gel strips are retained in the small chip. Note that due to known chip defects, Figure 3C The isolated empty flow channels in the shown small chip never received gel, meaning the small chip achieved 49 out of 49 successes.

[0061] Optimization schemes for minimizing noodling and maximizing manufacturing yield during peeling include the following steps: (1) Before flowing, seal the microfluidic chip to be filled with gel on a smooth non - adsorbent flow substrate; (2) Fill the ROI of the chip with gel at 0 - 4 °C and cool the chip after 15 minutes; (3) Allow condensate to clear from the chip and / or flow surface by introducing the assembly to a temperature of about 23 °C for 0.5 - 1 minute, 1 - 5 minutes, 1 - 15 minutes, or 1 - 60 minutes (depending on the composition of the carrier gel material and reagent used); (4) Peel the flow substrate from the chip (or peel the chip from the flow substrate); (5) Examine the chip in a microscope for areas of noodling, areas without barcoded gel, or areas containing overlapping barcoded gel (similar to clogging and leakage); (6) Discard the chip if there are irregularities above a specific quality threshold. If not, store the chip in a durable manner (possibly within a chip cassette) and bin by quality.

[0062] In some embodiments, the small chip is stored with its initial flow substrate intact.

[0063] In some embodiments, the flow substrate is removed from the small chip before storage, and the small chip is protected from debris with a chip cassette and / or laminate.

[0064] In some embodiments, if the small chip is to be stored in a humid environment, enclose the small chip with a moisturizer in a sealed system such as a bag or chip cassette.

[0065] In some embodiments, dehydrate and store the small chip by storing it in a dry environment or by first freeze - drying and then gelling (lyophilizing).

[0066] In some embodiments, the small chip is stored at - 20 °C. In other embodiments, the small chip is stored at 4 °C. In some embodiments, the small chip is stored at room temperature. In some embodiments, the small chip is stored in the temperature range from - 20 °C to room temperature.

[0067] Figure 4A and Figure 4B Shows a view of an exemplary chip cassette 402 for storing a single small chip.

[0068] The chip cartridge is designed with a movable hinge 403, so there are cavities on both sides of the chip cartridge. However, the chip cartridge can be designed to accommodate any number of small chips (e.g., 2, 4, 8, or some other even number that may be convenient for the end user). Each chip cartridge can also be packaged in a larger box / case, and then the larger box / case is also sealed to make it waterproof and airtight. It should be noted that the movable hinge 403 is not necessary for the design work; instead, it is a cost-reducing feature (one molded part instead of multiple molded parts). If needed, the design of the chip cartridge can also be more similar to a traditional two-piece storage container (e.g., a food storage container). At a high level, the design of the chip cartridge is intended to provide a closed environment for the storage of one or more small chips, facilitate the user to unpack and use the small chips stored therein, and at the same time reduce the risk of damage or deterioration.

[0069] Figure 4A An exploded view of an exemplary chip cartridge 402 is shown, where the environmental seal or gasket 404 is removed. Figure 4A A small chip 406 with an exemplary channel 408 is also depicted, and the channel is filled with a reagent dissolved in a gel carrier material (e.g., hydrogel or sol-gel). It should be noted that the size of the channel 408 is enlarged here only for exemplary purposes. Generally, a small chip will include 25 to 300 channels. A protective layer 410 is shown to protect the material contained in the channel 408 until the small chip 406 is ready to be used. The protective layer can take many forms, including a PET or glass layer. Figure 4A A moisturizer 412 is also shown, which is configured to fit within a recess 414 of the chip cartridge 402 and prevent the material in the channels 408 of the small chip 406 from drying out. Figure 4B A small chip 406 disposed within the chip cartridge 402 and a gasket 404 positioned in place are shown, and the gasket prevents air from entering the chip cartridge 402 after the chip cartridge 402 is closed.

[0070] The flow-based DBiT process requires the use of two different types of chips, chip A and chip B. Their only difference lies in the orientation of the channels in the active area. Sequentially applying and flowing through chip A and chip B can generate the orthogonal cross-flow pattern unique to DBiT.

[0071] Figure 5Shows the active and non-active regions of the DBiT-seq microfluidic device. The region within the small orange square 502 is active, and all other regions are non-active. After filling the chip with the barcoded gel and curing the gel, all regions outside the square 502 can be cut off and discarded as waste. This enables the fabrication of A-orientation and B-orientation chips on the same chip design, without the need for two separate production lines as is currently the case. Note that if each inlet were fully filled and flowed to the outlet side, how much barcode would be wasted; for every 10 microliters of barcode mixture loaded into the chip, only about 20 nanoliters (or 1 / 5000 of the loaded barcode) are needed to fill the active region. We will later see how to take advantage of this fact to reduce barcode waste. In some embodiments, the microfluidic chip can be specifically designed to prepare multiple small chips from a single microfluidic chip (see Figure 11 , Figure 13 and Figure 15 ).

[0072] The following are some of the main failure modes encountered when using our preferred chip filling method. The incidence of each failure mode depends on the manufacturing method selected. The phenomenon of delamination will be described below and can be minimized by choosing a suitable flow substrate and enhancing the affinity of the gel for the microfluidic channels. By using a microfluidic chip without any sharp corners and removing all debris and other sharp surfaces that could act as bubble generators, the formation of bubbles within the gel during the flow in the filling stage can be minimized. Condensation may occur after the small chip is peeled off when the small chip is below room temperature and / or in a very humid environment. Condensation usually results in the mixing of active components between channels. Condensation can be avoided by peeling off the small chip only at room temperature or near room temperature and in a humidity-controlled environment. The last failure mode is more subtle and usually occurs when preparing the chip with a shorter flow time. This can lead to a change in the concentration of active components between channels in the active region of the microfluidic device. Due to the concentration change, the imprinting of the small chip is uneven, which may in turn lead to excessive technical variation in downstream analysis. Uneven loading can be identified by conjugating a fluorescent molecule (i.e., a fluorescent dye) to the active component, and this conjugation can be used in the chip to be used as a stamp, as well as in a test chip under the same flow conditions as the chip to be used as a stamp. To avoid uneven loading, allow the gel to flow for a sufficient length of time (2 to 10 minutes in most configurations) such that the concentrations asymptote to their equilibrium values (i.e., the concentrations of the active components in the active region and the inlet should be as close as possible).

[0073] When manufacturing a microchip by filling the chip with a gel, the choice of the flow substrate can have a significant impact on the results at least in the following aspects: (1) How effective is the passive seal between the microfluidic chip and the flow substrate? The better they seal without clamping, the easier it is mechanically to fill the chip with the gel without any leakage (referring to the improper entry of the gel from one flow channel into another). Although a flow substrate with poor passive sealing with the microfluidic chip can be used, the two must be clamped tightly to prevent leakage. (2) What is the anti-flow ability of the flow substrate material? When using a flow substrate with extremely strong surface hydrophobicity, a greater vacuum pressure may be required to overcome the repulsive force of the immersed liquid gel against the coated hydrophobic surface when aspirating the liquid gel embedded with reagents through the microfluidic chip channels. Therefore, using a flow substrate with extremely strong surface hydrophobicity also promotes the formation of air bubbles. (3) How difficult is it to peel the microchip from the flow substrate after the flow (or, in some cases, peel the flexible flow substrate from the microchip) without tearing the gel strip out of the microchip channels? Materials that bind well to the gel (such as uncoated glass) tend to result in more "noodling" (for example, see Figure 3A and Figure 3B ). This reduces the manufacturing yield and increases the effective unit price. (4) How expensive is the flow substrate? An expensive substrate increases the unit price.

[0074] Given the above considerations, several tested flow substrates are listed below, arranged in descending order of peelability and flow success rate. (1) Polycarbonate (PC) or polyethylene terephthalate (PET) sheets with a thickness between 100 and 500 μm. The plastic surface is not easily adhered to by the gel, can be passively bonded with PDMS for flow, and has moderate hydrophobicity. The flexibility of the thin sheet enables the substrate to be peeled from the microchip at a very obtuse angle (almost 180 degrees), which helps the gel strip stay inside the microchip. (2) A glass slide coated with PFOCTS (1H, 1H, 2H, 2H-perfluorooctyl-trichlorosilane), manufactured by vapor deposition in a vacuum chamber (vented to a chemical hood). (3) A glass slide coated with RainX, a commercial spray coating made by our laboratory by repeatedly spraying and wiping blank glass slides with Kimwipes.

[0075] In some cases, a variety of other materials can also be used as flow substrates and can be used for certain carrier gel materials. The following materials have been tested as flow substrates: glass slides coated with poly-L-lysine, silanized wafers, aluminum, polystyrene, thin-film PDMS, FEP (fluorinated ethylene propylene) film, polyetheretherketone (PEEK), high-density polyethylene (HDPE), thin-film polytetrafluoroethylene (PTFE), and polyimide (PI) have been tested as flow substrates. Other candidate flow substrate materials include: cellulose, PP, and PLA.

[0076] An exemplary DBiT process can be performed by an end user using two stored microchips. The process includes the following steps: (1) Unpack the first stored microchip. The unpacking process may include a controlled thawing procedure to avoid premature liquefaction of the gel embedding the barcodes or condensation deposition on the active surface of the first stored microchip (resulting in interference of the barcodes with each other through the diffusion of condensed water droplets). (2) After preparing the tissue sample, now also deposit 100 to 200 microliters of ligation buffer on the tissue sample. (3) Incubate the buffer on the tissue sample at room temperature for 15 minutes. (4) Pour off the excess buffer, but do not allow the tissue sample to dry completely. (5) Place the microchip on the tissue at the desired position and orientation. (6) Clamp the microchip down onto the tissue sample. (7) Control the temperature of the microchip and the tissue sample during a fixed incubation time while allowing the gel embedding the barcodes to interact with the tissue sample. (8) Remove the microchip from the tissue sample after the desired incubation period (e.g., 2, 4, or 16 to 24 hours). (9) Wash the tissue in warm wash buffer (37 °C) to remove excess remaining gel. (10) Repeat each of the foregoing steps for the second stored microchip. The second stored microchip will typically be placed in the same position as the placement position of the first microchip, with its orientation such that the channels of the second microchip are orthogonal to the orientation of the channels of the first microchip.

[0077] In some embodiments, the ligation buffer is added to the pre-loaded microchip. Thus, steps 2 to 4 are optional steps.

[0078] In some embodiments, the microchip is not clamped onto the tissue. Instead, lateral diffusion within the tissue is allowed. We have conducted similar tests, and for uniformly dense regions of interest, the diffusion rates between channels are approximately equal; thereby increasing the labeled area of the barcodes by one channel wall thickness. The result is that there is little dead space between the channels; while dead space typically accompanies the channel walls. Based on our experiments, the barcodes diffuse into the analyte and hybridize mainly to those regions not occupied by pre-hybridized / ligated barcodes.

[0079] The following description applies to the above - described exemplary DBiT process: (1) Since there is no flow and only imprinting, there is no risk of flow failure. (2) One way to precisely locate the region of interest for placing the second smallest chip is to use fluorescently conjugated bovine serum albumin (BSA), where one color is in channel 1 (e.g., green, using FAM or a similarly colored fluorescent dye conjugated to BSA), and the other color is in the last channel (e.g., red, using Cy3 or a similarly colored fluorescent dye conjugated to BSA). Any colorant can be used to label the outer - edge flow channels, but BSA has the advantageous property of non - specifically binding to a variety of biological agents, including tissue components, common substrates, and poly - L - lysine (PLL), which is a common biological coating applied to the slide surface to facilitate the binding between the slide and the tissue fixed thereon. Even if part or all of the outer - edge flow channels do not pass through the tissue, they will still deposit a recognizable colored line on the flow substrate, thus successfully delineating the active area of the applied small chip. (3) The clamping pressure is consistent with the conventional DBiT process, which means that any clamping device suitable for conventional DBiT can be used to fix the small chip. (4) At room temperature or colder temperatures, using the described embodiment may allow for a longer ligation time, but this is not the case with conventional DBiT. This is due to the fact that the gel always remains solid, and the barcodes are transferred through diffusion from the gel matrix to the tissue. We have observed that even during an overnight ligation reaction, the barcode diffusion between channels is extremely limited, which means that a long and slow ligation reaction can now be achieved. However, in conventional DBiT chips, chips using the passive common - outlet technique are not recommended for incubation longer than 1 hour because as the reaction time lengthens, the retention of reagents within the chip leads to a significant increase in the frequency of inter - channel diffusion.

[0080] One interesting point is to determine the comparison result of the quality of the downstream sequencing data generated by the described embodiment with the data generated by the standard flow - through method. Given that the non - spatial barcoding steps between the two methods are similar, we considered whether imprinting might transfer sufficient active components (in the case of DBiT, oligonucleotides act as different combinatorial spatial barcodes for downstream NGS) to the region of interest (ROI) in the tissue.

[0081] Compared with the conventional DBiT operation where the barcoding reagent flows through the tissue, the small - chip - based DBiT treatment exposes the tissue to fewer reagents. The DBiT chip inlet can accommodate up to 10 μL of liquid, and in principle, the entire 10 μL could flow through the tissue, exposing the tissue to the entire volume of an aqueous solution containing a very large amount of active components. For example, a 10 - μL solution of 5 micromoles contains 3x10 13oligonucleotides. In practice, typically about 2 to 5 μL are delivered to the outlet of each channel, but even 10% - 50% of this amount is a very large number of oligonucleotides.

[0082] In contrast, the described embodiments do not move liquid along the flow channels; rather, only the gel volume directly above the region of interest (ROI) can deliver the active ingredient to the ROI. Assume that for a 10 μm chip, the channel length in the ROI is approximately 5 mm. The volume of this channel is approximately 10 μm × 10 μm × 5000 μm = 0.5 nanoliters. For a 5 μM oligonucleotide solution, this yields "only" approximately 1.5 billion oligonucleotides in each flow channel. Assuming a cell spacing of 10 μm, 5000 μm spans approximately 500 cells. This means that each cell can receive up to approximately 3 million oligonucleotides (some oligonucleotides may not flow out of the gel and into the tissue).

[0083] Is this sufficient? For a rough comparison, in a spatial ATAC-seq run, the number of fragments we typically recover is about 50,000 to 100,000 labeled gDNA fragments per tixel in a 25 μm run. For spatial whole-transcriptome sequencing, we recover 3000 to 5000 UMIs per tixel. Table (1) below summarizes the calculation methods discussed in this article and lists the key parameters that can be used to calculate the available amount of oligonucleotides per analyte in spatial ATAC-seq and spatial whole-transcriptome detection of the DBiT-seq technology at a specified chip resolution.

[0084]

[0085] Table (1)

[0086] Based on the above table, in the 10 μm configuration, there are more than 5000 available barcodes per UMI, and in the 25 μm configuration, there are more than 9000 available barcodes per UMI. Similarly, for each fragment in the 10 μm and 25 μm configurations, there are more than 200 and 600 available barcodes, respectively. Although this ratio is much lower than that of the flow-through tissue method, we did not observe a quality decline in the small-chip-based DBiT shown in this article (e.g., Figure 7 to Figures 9 and 20 to Figure 24 ) Figure 24It also includes co - analyzing ATAC and WT samples, which further reduces the number of oligonucleotides available for each analyte: to 197 [5.12E+06 / (25000 + 1000)] and 588 [4.70E+07 / (75000 + 5000)] at 10 - micron and 25 - micron chip configurations, respectively. The number of available oligonucleotides can be easily increased by increasing the height of the microfluidic channels or simply adding a higher concentration of oligonucleotides in the gel.

[0087] The presented small - chip - based DBiT processing requires soaking the tissue with approximately 120 μL of ligation buffer (a mixture of T4 ligase buffer, water, NEBuffer, T4 DNA ligase, and Triton X - 100) before placing the small chip on the tissue. This provides an abundance of T4 ligase in a non - spatially - controlled manner. Since the enzyme is the same in all flow channels, it does not need to be delivered in a spatially - controlled manner. And since the ligase is a protein complex (about 80,000 daltons), which is larger than the oligonucleotides (our barcodes are about 68 to 75 nucleotides (nt), or about 20,000 daltons), it is more likely to form a concentration gradient along the length of the flow channel. This innovative method of bulk - providing ligase has been shown to be compatible with the standard DBiT protocol.

[0088] Data Quality Comparison between Small Chip-based DBiT Processing and Standard DBiT Processing

[0089] To demonstrate the feasibility of delivering active ingredients to the target substrate using small - chip - based DBiT processing, we conducted three consecutive studies. (1) Active ingredient delivery. First, we attempted to determine whether the small chip could deliver fluorescently - labeled oligonucleotide barcodes to pre - prepared tissue sections. (2) Adjusting process parameters for spatial ATAC - seq. Next, we performed some complete spatial ATAC - seq DBiT experiments using small - chip - based DBiT processing instead of the standard flow method and compared the data with the body of data derived from standard spatial ATAC - seq experiments on similar (but not matched) tissue samples. Using this data (outlined below), we adjusted some processing parameters of the small - chip - based DBiT processing method, including (but not limited to): a. Stripping conditions; b. Ligation incubation time and temperature; c. Composition of the ligation buffer, including salt content. (3) Head - to - head comparison. Third, using the optimized processing parameters derived from the second - round experiments, we performed a head - to - head comparison of two standard DBiT - seq and two small - chip - based DBiT - seq techniques on mouse cerebellum tissue, which was snap - frozen and embedded in optimal cutting temperature compound (OCT).

[0090] Figures 6A to 6BShows the delivery of fluorescently labeled oligonucleotides to two different fresh-frozen mouse embryo sections. The sections were thawed, fixed with 4% PFA, and permeabilized and reverse transcribed to generate cDNA (complementary DNA) using cytoplasmic mRNA (messenger RNA) as a template. The resulting initiation sites were used as target sites and hybridized and blunt-end ligated (by T4 ligase) with ssDNA (single-stranded DNA) barcodes via a fluorescently labeled oligonucleotide linker (green bar graph, FAM tag). A small chip was used to deliver the labeled oligonucleotides and linkers. After the A-labeling and ligation incubation, the tissue was washed with warm NEB 3.1 buffer, and then orthogonally stamped, incubated, and washed with a similarly labeled linker and oligonucleotide, where the array was oriented in the orthogonal direction (red rows, Cy3 fluorophore), but this time the oligonucleotides hybridized and ligated with the previous oligonucleotides rather than with the cDNA targets. Figure 6A The ligation condition in Figure 6A was incubation at room temperature for 2 hours, Figure 6B and the ligation condition in Figure 6B was incubation at 37 °C for 2 hours. Given that Figure 4A the incidence of non-specific binding was lower under the room temperature incubation condition in Figure 4A , this condition was preferred in subsequent optimization tests.

[0091] Using the above method, we set out to demonstrate the feasibility of performing complete spatial ATAC-seq and spatial whole transcriptome seq (spatial WT-seq) by microchip stamping rather than flow barcoding. Based on the successful results of approximately 12 DBiTs using our microchip, our preferred protocol uses the following process parameters: (1) the same barcode concentration as standard DBiT; (2) ligation at 16 °C for approximately 16 hours, or ligation at room temperature for 10 minutes followed by ligation at 37 °C for at least 2 hours; (3) using a ligation buffer with the same composition as standard DBiT, except that the buffer was soaked on the tissue before stamping (instead of being delivered through a microfluidic channel as in standard DBiT).

[0092] Using the above processing parameters, we set out to directly compare the performance of microchip-based DBiT processing with standard DBiT processing on the same day, and used four sections from the cerebellar tissue sample of the same mouse, which were cut and mounted on the same day. The sections were stored at -80 °C until the day of the experiment, when thawing and fixation were completed by the same technician, and then pre-digested using Tn5 transposase diluted from the same master batch with barcodes and a ligation buffer prepared by the same technician according to the same formula on the same day. All of these represent our best attempt to eliminate any differences between samples, except for the experimental conditions, i.e., two of the four runs were performed using the standard flow protocol in a 25-um resolution single-layer microfluidic chip, while the other two runs were performed using a 25-um resolution microchip.

[0093] Although longer ligation incubations generally produce better microchip-based processing results than shorter ones, we chose to perform a uniform ligation (10 minutes at room temperature followed by 30 minutes at 37°C) in all four runs to standardize the temperature and time between the two conditions and thus best discern any differences in barcode delivery efficiency between the methods.

[0094] The performance of the four runs was roughly comparable, except that the standard flow runs showed slightly less variation related to the number of rows and / or columns. This indicates that at the same concentration and the same ligation temperature and time, the fragment recovery of FlowGel (microchip-based processing) was slightly lower than that of standard DBiT. Nevertheless, according to most metrics, these runs were not statistically distinguishable at N = 2 replicates. To reduce the ratio of artifacts in row / column alignment of fragment counts, the barcode concentration in the carrier gel of the microchip can be increased, and the ligation temperature and ligation time (i.e., 2 hours, 4 hours, or 16 hours at room temperature) can be optimized for this technique.

[0095] Figure 7 Four electrophoretograms are shown resulting from a head-to-head comparison of two sets of conventional spatial ATAC-seq runs (D00975 / D00976) with two sets of matched microchip-based spatial ATAC-seq runs (D00977 / D00978). Figure 7 The fragment length distribution for each of these four runs is shown, where the nucleosome-free peak (between 270 and 425 base pairs) and its subsequent peaks spaced 147 base pairs apart showed good agreement with the expected ATAC-seq signal in all four runs. In Figure 8C and Figure 8D the microchip-based runs shown, the separation of the mono-nucleosome (270 + 1*147) and di-nucleosome (270 + 2*147) peaks was better.

[0096] Figures 8A to 8D Fragment count TIXEL maps are shown obtained from a head-to-head comparison of two matched flow-based spatial ATAC sequencing runs ( Figures 8A to 8B ) and two matched microchip-based runs ( Figures 8C to 8D ). Although the microchip-based runs showed a higher degree of artifacts related to inter-channel differences in barcode delivery, all four runs showed similar homology between fragment counts and tissue morphology. It should be noted that each square in the TIXEL map of the microchip-based runs corresponds to the intersection of a channel of the first microchip with a channel of the second microchip, where the channels of the second microchip are orthogonal to those of the first microchip as described above.

[0097] Figure 9AShows a comparison sequencing statistics data table with matching flow-based runs and chiplet-based runs. Definitions: #Cycles = The number of additional PCR cycles determined by qPCR amplification of aliquots of the library. The total number of amplification cycles equals 5 + the number of additional cycles. % Duplicates = The number of sequencing reads that are duplicates of each other (higher values indicate deeper sequencing and / or lower library diversity). FRIP = The number of read fragments in the peak (higher fractions are better). TSS = Transcription start site enhancement score (higher scores are better). Npeaks = The number of peaks found in the gene locus. The average number of fragments per TIXEL represents the average number of fragments recovered per TIXEL on each tissue. The values in the bottom two rows ("Good" and "At Risk") represent the thresholds of the normalized sequencing metric scoring criteria. Relative values indicate whether higher or lower values are desired in each category. For each run, the metrics were within the parameters used by those skilled in the art of chromatin analysis, except for runs D00973 and D00975, which showed an unusually high number of non-nuclear reads. This may indicate that flow-related factors (e.g., the vacuum pressure applied to the channels when in contact with the tissue) can disrupt compartments filled with non-nuclear genomic DNA sources (e.g., mitochondria), while chip imprinting does not (since no vacuum is applied to the chiplets when in contact with the tissue).

[0098] Figures 9B to 9E Shows the results of the chiplet-based DBiT run. Figure 9B Describes the fragment recovery rates of genomic regions associated with the genes Apol7d, Olfr807, Hist1h2ak, and Acrv1. Notably, the spatial distribution of these markers was completely undisturbed by technical artifacts, and their distribution patterns highly corresponded to the tissue morphological structures visible under bright-field microscopy.

[0099] Figure 9C Shows the results of unsupervised spatial clustering that groups TIXELs (tissue elements) according to differences in the genomic maps of the recovered fragments. The upper figure shows a UMAP (Uniform Manifold Approximation and Projection) plot corresponding to this clustering scheme. This way of representing data in a reduced-dimensional space is well-known to those skilled in the art, and the better the separation between TIXELs in this representation, the richer the dataset.

[0100] Figure 9D Shows an electropherogram of the Agilent TapeStation, which shows the fragment length distribution of the fragment library generated after sequencing Figure 9B and Figure 9C image. The first large peak at 270 - 400 base pairs is consistent with the typical separation between histones in chromatin, indicating that meaningful tagging by the Tn5 transposase occurred in the tissue sample.

[0101] Figure 9EIt also depicts the TSS (transcription start site) enhancement score and the number of recovered fragments in each TIXEL. A good distribution roundness indicates a higher library quality. The population with a slightly lower TSS score (below TSS 8) represents regions with lower chromatin accessibility, shown as cluster 4 in Figure 9C as cluster 4.

[0102] It should be noted that the microchip-based processing method described herein can significantly save the workflow in terms of the time required for laboratory technicians to perform DBiT processing. Practical tests have shown that the actual operation time of technicians can be reduced by two-thirds. For example, in the test, the actual operation time of technicians using a 25-um resolution decreased from approximately 2.2 hours to 0.6 hours. It should be noted that due to the increased connection time, using microchip-based processing can still lead to an increase in the total time required to perform DBiT processing. However, the reduction in actual operation time enables technicians to focus on other tasks, such as performing more microchip-based DBiT-seq while waiting for the connection to complete.

[0103] Performance Improvement (ROI and Resolution)

[0104] Since the flow processing of microchip-based processing occurs a) without endangering the tissue and b) on a known flat substrate, longer flow paths can be achieved without the risk of flow irregularities or tissue damage. Since the flow during microchip creation does not need to consider substrate heterogeneity or non-smoothness, microchip-based processing is easier to improve the resolution than flow-based processing. At a resolution as low as 5-um channel width, the flow proceeds very smoothly. In contrast, the flow on tissue encounters serious problems starting from a channel width of approximately 10 um. This enables spatial analysis with a spatial resolution as low as 5 um or lower using FlowGel.

[0105] Unfortunately, when reducing the channel width to improve the spatial resolution while keeping the number of channels unchanged, it has a side effect of reducing the field of view. From a biological perspective, this increases the spatial bias when selecting regions of interest. Specifically, a typical 50-channel chip used in DBiT reduces its active area from 2.5×2.5 mm at 25-um resolution to 1×1 mm at 10-um resolution, and then further reduces the active area to 0.5×0.5 mm at 5-um resolution. These fields of view are too small for many end users, so expanding the active area of the chip will have great practical significance in this field.

[0106] Another side effect of reducing the width of the channels in the ROI is that the amount of barcodes near the ROI (and that can diffuse into the tissue within the ROI) will be reduced. For example, compared to a cross-sectional area of 25×25 = 625 square microns in a typical 25um channel, a 10×15um channel has a cross-sectional area of 150 square microns. This reduction in area results in a reduction in volume, and thus the number of oligonucleotides per target tissue element (“tixel”) is reduced by 76%. This can be slightly improved by increasing the height of the channels in the high-resolution chip (e.g., from 10×15um to 10×17.5um). This serves a dual purpose: 1) during stripping, the taller channels will accommodate more gel (more contact area with PDMS compared to the flow substrate), and 2) the cross-sectional area in the ROI will increase from 150 square microns to 175 square microns, or only a 72% reduction compared to the 25um resolution chip.

[0107] Although this may sound like a significant reduction, the area under the channels is also reduced, and thus the number of targets requiring barcodes is reduced (proportional to the area of the tissue element or tixel). For example, the lateral area of a 25um tixel is 625 square microns, while the lateral area of a 10um tixel is 100 square microns (lateral here refers to in the plane of the tissue section).

[0108] Using small-chip-based processing, the problem of reduced field of view can be improved in the following ways: (1) adding more channels to expand the small chip; (2) placing multiple small chips on the same substrate.

[0109] The disadvantage of adding more channels is that additional space must be allocated on the chip for the inlets and outlets to provide docking ports for the additional channels. The inlets dock with liquid loading tools (e.g., single-channel or multi-channel pipettes), while the outlets dock with a vacuum that provides a pressure gradient to facilitate flow.

[0110] One way to reduce the additional space requirements at the outlets is to merge the flow channels after they pass through the active area; this strategy is referred to below as the “common outlet” scheme. This saves space by reducing the number of separate outlets present in the chip. But it also introduces a contamination-type failure mode: if the active components mix after the corresponding flow channels merge, and subsequently these mixed active components flow, diffuse, or otherwise back-diffuse into the active area, the chip may not be able to confine each active component to its intended target delivery area on the substrate.

[0111] Figure 10A “superchip” is shown, which is characterized by 288 independent inlets. Each of these 288 inlets feeds into a channel that passes through the active region. After passing through the active region, these channels merge into two groups, each of which terminates at a common outlet. By omitting 208 of the 210 possible outlets, the space saved is used to add more channels, thus achieving a larger field of view. For this particular superchip with a channel width of 10 um and a pitch of 15 um, the width of the active region is 288×(.010 +.015) = 7.2 mm. If two such chips are cross-flowed or cross-imprinted to create a DBiT region of interest, this will result in a field of view of 7.2×7.2 mm or 51.8 square millimeters, which is 51.8 times the current DBiT 10 um field of view (1×1 mm).

[0112] Since only the active region (represented by square 1002) needs to be imprinted onto the tissue, it can be separated from the rest of the chip (forming a small chip set), stored, and delivered to the end user. The same chip can be filled with different barcoded gels (e.g., a set of B barcodes instead of A barcodes), then split into small chips, and then imprinted onto a substrate (which has already been imprinted with a series of barcodes by A small chips), with the orientation rotated relative to the previously imprinted array (such as rotated 90 degrees or any other selected angle), thus enabling full-function DBiT analysis with a very large field of view at 10 um resolution.

[0113] This chip can in principle be used to implement standard flow DBiT. However, this presents two problems. First, in order to achieve cross-flow, the chip must be fixed to the substrate in two directions, and existing hardware cannot fix such a large-sized chip in two different directions on the same substrate. Second, due to the active region with a large number of fine channels, a single-layer chip with so many channels will experience a large amount of flow irregularities at 10 um resolution. The high flow resistance due to the slender channels causes serious problems, requires high vacuum pressure, and may lead to more blockages or cross-channels than usually smaller chips. Third, during the connection time (about 0.25 hours - 24 hours) required by most DBiT protocols, the reagents will mix at the common outlet and back-propagate into the active region, resulting in the channels being contaminated with the wrong barcodes and reducing the spatial fidelity of the analysis. Methods for reducing this back-propagation include using valves to limit backflow or diffusion, and evacuating while incubating.

[0114] When used to produce microchips for chiplet-based processing, the chip design has none of these problems when filling the chiplets and when imprinting the chiplets on tissue. First, the chiplets can be cut from the rest of the chip and placed in any orientation or manner desired by the end user. Second, since there are no uneven points that need to be sealed, there is no need to clamp the chip on a flowing substrate during the filling process. As a result, flow irregularities are almost non-existent, and even if they do exist, these deformed chiplets can be discarded before contacting the tissue. During imprinting, the viscosity of the gel at room temperature or colder connections inhibits the backpropagation of the barcode from the common outlet to the active area.

[0115] In addition, this type of chip is easier to fabricate and has lower operating costs than current DBiT bilayer chip designs (such as those described in US 63 / 328,195 “MICROFLUIDIC CHIP”). It is a single-layer chip and is thus easier to fabricate than a bilayer chip. A bilayer chip requires the two layers to be fabricated separately and then aligned, bonded, and visually inspected (whereas a single-layer chip does not require alignment and bonding).

[0116] Finally, compared to the two production lines for Type A and Type B bilayer flow chips, the Type A and Type B chips do not require a separate production line. This significantly saves the cost of fabricating the superchips and filling the chips compared to fabricating the bilayer A and B chips and flowing them on tissue. In summary, chiplet-based processing enables a more dense chip design than standard flow-based methods, and this type of chip with such a large active area will enable researchers who desire single-cell resolution analysis of target analytes to do so with a smaller spatial deviation while capturing a larger proportion of the tissue samples of interest.

[0117] Figure 11An image of a microfluidic chip 1100 with a mother chip design configured to fabricate multiple microchips is shown. The microfluidic chip 1100 has a channel resolution of 50 um in the active region. Each well accommodates 5 ul of carrier gel. The common outlet (the upper right well) is far enough from the active region (depicted by the dashed square) such that any mixing of the carrier gel does not contaminate the active region. After flowing at 37 °C, the entire device is cooled to room temperature to fix the gel in place. Before peeling, a cryogenic blade is used to cut the microchips to test whether the cutting process would contaminate the ends (bottom) of the microchips. This did not occur, demonstrating the feasibility of creating many microchips from one mother chip. This design can create up to 12 active regions of 4 mm × 4 mm in one flow, meaning the cost per active region will be 1 / 12 of a 50-channel bilayer DBiT active region chip. This also significantly reduces the oligonucleotide cost per active region; from 6 uL / channel to 5 uL / channel / 12 chips. The manufacturing cost of this serpentine chip is roughly the same as that of current single-layer chips, at approximately $100. Meanwhile, the current cost of bilayer chips is approximately $250. Since each chip supports 12 runs, this reduces the microfluidic chip cost from $2 × 250 = $500 per run to $2 × 8.33 = $16.66 per run. This chip is much more difficult to execute in flow-based DBiT processing because all active regions need to be created continuously on the tissue, increasing the likelihood of flow irregularities, and each blockage or leak point will obscure or contaminate the TIXELs to be formed downstream. This becomes even more important as the channel size is reduced to market-relevant resolutions (i.e., 10 um or less).

[0118] In some use cases, the end user prefers to have multiple fields of view on the same substrate rather than maximizing the area of a single field of view. This is the case, for example, in tumor microarrays or other use cases where multiple different tissue sections need to be mounted on the same slide.

[0119] To address this use case, we can take advantage of the ability to separate the active regions of the microchips from the rest of the microfluidic device used to create the microchips and then place a pair of microchips (A and B) in each intended field of view. Below in Figure 12This situation is depicted. Each dashed line represents the field of view in which the end user wishes to perform spatial analysis. In this case, the tissue sections are evenly distributed on a 2×4 grid; however, in principle, it is not necessary for the small chips to be arranged on a regular grid. This design greatly reduces the workload of tissue technicians - they would otherwise need to precisely cut and position eight or more ultra-thin tissue sections onto predefined microzones on a single substrate; the technician can try to leave enough space between the sections to place the small chips, and then the exact position (and rotation) of the small chips can be selected at runtime to fit the position of the sections.

[0120] Figure 12 The configuration shown is a common way to mount multiple tissue sections on a single slide (e.g., for tumor microarrays). Tissue technicians using small-chip-based processing no longer need to precisely mount these tissue sections to predefined positions, as the number, rotation angle, and position of the small chips are all selected by the end user at runtime.

[0121] After performing imprints A and B in sequence, each field of view now has 210×210 = 44,100 TIXELs, each with a different A and B barcode. Typically, the end user needs to lyse eight fields of view separately and then use different primers during next-generation sequencing library preparation so that the targets in different fields of view are not confused during downstream sample pooling.

[0122] However, small-chip-based processing enables an easier method. Each of the eight A small chips contains a different set of 50 A barcodes (e.g., A1 - A50, A51 - A100, A101 - A150, A151 - A200, A201 - A250, A251 - A300, A301 - A350, and A351 - A400). At the same time, each of the eight identical B small chips has only one set of B barcodes. Now assume that all fields of view are digested in the same lysis buffer after imprints A and B. Then the resulting lysate pool undergoes the same purification and library amplification steps and is sequenced. This will result in 8×210×210 = 8×44,100 = 352,800 TIXEL units in the downstream analysis, where groups of 44,100 TIXEL units each correspond to the appropriate regions of each tissue sample, as designed in US 63 / 252091, METHODS AND DEVICES FOR SPATIALLY ENCODED BIOLOGICAL ASSAYS. However, this method is superior to the method described in that patent for the following reasons.

[0123] To facilitate handling and placement of the small chips during manufacturing and final use, each small chip may include grippable features. For example, a small segment of wire can be suspended in liquid PDMS and retained during curing such that one end of the wire is embedded in the region of the resulting microfluidic chip configured to be removed to form the small chip. This works as long as the wire is fixed above the contact layer, thus avoiding interference with active ingredient delivery. While an example of a wire is provided, other grippable features are also considered within the scope of the described embodiments (including but not limited to surface tension adhesion between the back side of the small chip and a temporary support and / or a clamping bar). As the number and / or density of small chips increases, this type of feature will become increasingly important as it will be difficult to pick up densely spaced small chips from the side without disturbing adjacent small chips.

[0124] Standard DBiT requires very thin tissue sections (with a thickness between 5 μm and 10 μm) to create a successful cross - flow across the tissue. Since chip - based processing does not flow through the tissue, it does not require thin tissue sections. In principle, there is no upper limit to the tissue thickness, and barcoded gels can even be imprinted onto non - planar sections. For example, flexible PDMS chips can be imprinted on the surface of a whole organ (rather than an organ section). This enables two important embodiments.

[0125] Vibratome - sectioned DBiT. Since thick tissue sections are opaque, vibratome sections (about 40 μm thick or thicker) cannot be used for most microscopy techniques. However, chips can be used to imprint barcodes on top of the sections regardless of the section thickness. This will provide spatial omics or other barcode - driven analytical methods for those who cannot use cryosectioning machines, thus helping to popularize spatial omics.

[0126] Three - dimensional DBiT. Consider a whole intact organ, such as a mouse brain. Cut it in half to form a slightly flat surface at the incision. Apply the A&B chips to this flat surface. After barcode transfer, cut off the barcoded top layer of the brain (e.g., the outermost 50 microns), and then repeat this process on the newly exposed flat surface. Repeat the operation until the entire brain has been barcoded in this way. Combining two - dimensional omics maps with computer simulation, a three - dimensional map of the brain can be generated with high throughput and low cost. The best tissue form for this method is tissue fixed by perfusion to avoid fixing each section individually.

[0127] DBiT of tissue sections suspended in a matrix. Suspending tissue sections in a gel matrix or other matrix (e.g., for tissue clearing or expansion microscopy) has proven beneficial. This will enable existing microfluidic technologies to achieve higher sensitivity and / or higher-resolution spatial analysis. For example, a 10-μm resolution barcode array imprinted on a 5-fold expanded section has the same resolution on the expanded tissue as a 2-μm resolution chip, which is subcellular in many tissue types. The expanded tissue will be suspended in one of many polymer gels. To make the tissue thin enough to be compatible with standard DBiT, the embedded tissue may need to be re-sectioned after embedding and / or expansion, where a 10-μm thick section contains only about 2-μm thickness of the pre-expanded target, allowing standard DBiT to recover sufficient information. For example, in the resulting thin sections, cell nuclei may be missing or only partially intact, reducing the quality of epigenetic analysis in these regions of the tissue. Chip-based processing without re-sectioning makes it easier to be compatible with tissue-embedding-based methods.

[0128] As previously referenced Figure 11 stated, to further reduce the manufacturing cost of the small chips, we can create a "master chip" that uses a set of inlets to supply flow to the downstream channels of multiple active regions. During our initial tests on a serpentine chip with 50-μm channels in the active region (as shown below Figure 13 ), no bubbles were found. We believe this is because the early versions of the chips had sharp corners or edges that interfered with the flow and promoted bubble formation.

[0129] Figure 13Shows a microfluidic chip 1300 with a serpentine design, which has 50 inlets (far right), 50-micron channels (channel-to-channel spacing of 30 microns), and a common outlet. The chip is made of PDMS, and the laminate is a polycarbonate film. The barcoded gels (even channels loaded with Cy3 conjugated to 50-base random ssDNA barcodes and odd channels loaded with FAM conjugated to 50-base random ssDNA barcodes) were kept molten on a heating plate during flow. After flowing in H2O at about 60 °C for 10 minutes, the assembly was cooled to room temperature and then placed in an unsealed Petri dish and stored overnight in a 4 °C refrigerator. The next day, the laminate was peeled off, and the entire chip was imaged for two-color fluorescence, and the results showed a very high uniformity of barcode concentration all the way to the delay loop (far left). Although all channels merge at the common outlet (yellow circle at the lower left), the backpropagation of contaminated barcodes only travels through a few millimeters, thus protecting the active area from barcode mixing (bottom inset). The chip consumes a total of 5 μL × 50 channels = 250 μL, which is half of the consumption (500 μL) of a standard single-layer DBiT chip per region of interest. Since the chip can be cut into approximately nine smaller chips, the oligonucleotide usage is only 1 / 18 of that of the standard DBiT, saving 94% of the barcode cost per ROI.

[0130] Figure 14 Shows Figure 13 The intensity map of the region within the dashed square in shows how the concentration of the active component of the flow channel (measured by fluorescence signal in this example) decreases near the distal end of the serpentine pattern, which is due to the inlets of some channels being closer to the active area than others. This concentration gradient can cause non-uniform deposition of the active component on the target substrate during imprinting, but this can be avoided by flowing the barcoded gel in the smaller chip for a longer time or at a higher pressure, thereby eliminating the chemical gradient change caused by the channel length difference.

[0131] Figure 15 Shows a microfluidic chip 1500 with another "mother chip" design, which shows how to fabricate a large number of smaller chips in a single microfluidic flow using a set of inlets and a microfluidic chip. After loading the inlets with the barcoded gel and flowing the liquid gel forward through the chip as much as possible, and after the gel is fixed in place, the smaller chips are separated from the rest of the mother chip using a blade. This process can generate dozens or hundreds of smaller chips simultaneously, thus significantly reducing the manufacturing cost and time. Since the entire mother chip can be first frozen, thawed, and then cut into smaller chips after peeling, the above process helps to reduce the time and cost associated with peeling.

[0132] Sacrificial Flow Substrate

[0133] Another variant of the described embodiment involves using a sacrificial flow substrate. The method using a sacrificial flow substrate will at least include the following steps: (1) Using a PVA film as the subsequent substrate, flowing the molten carrier gel through the microfluidic chip; (2) Cutting off the inactive area of the microfluidic chip; (3) Placing the small chip on a tissue section wetted with PBS and / or ligation buffer, where PBS and / or ligation buffer can dissolve the PVA film, thus enabling the reagents in the carrier gel to interact with the tissue section.

[0134] The advantage of this process is that it skips the peeling step, which has the risk of contaminating the gel strip inside the small chip before imprinting onto the target substrate.

[0135] The sacrificial layer can also be PVA, or any other film that will dissolve after contacting the target substrate, regardless of whether the target substrate is wetted or impregnated with a substance that a) will dissolve the sacrificial layer upon contact, and b) will not interfere with the intended use of the small chip for delivering the active ingredient. Additionally, the dissolving layer should not produce any by-products that may interfere with the analysis.

[0136] Combining A and B Barcoding Steps

[0137] Combining the A and B barcoding steps into one step (whether by flow or imprinting) will further improve the workflow. Chip-based processing provides a practical way to achieve such workflow improvements.

[0138] Consider two small chips. Now pair them up, perhaps adding some transfer buffer between them to wet the surface. Let them dock and incubate for a period of time. After the barcode diffusion exchange is completed between the two small chips, both small chips should now contain two sets of barcodes, and the overlapping positions of the A and B barcodes correspond to the desired tixels (tixel elements, tissue regions defined by the intersection of the A and B channels).

[0139] Then, the X small chip can generate available spatial data when docking with the tissue.

[0140] Figures 16A to 16BShows a configuration in which the tissue sample is located between the A microchip and the B microchip. The protocol for this method is as follows. Upstream chemical processing (tissue fixation, permeabilization, and reverse transcription or Tn5 tagging) can be performed on 8 or more tissue sections simultaneously in a 24-well plate, using a 200 to 500 μL reaction mixture (versus 800 to 1600 μL on a glass slide), and the washing is easier than on a glass slide. They can then be wetted with ligation buffer and T4 ligase (as in the sequential stamping microchip-based protocol described above), and sandwiched between the A and B microchips and incubated overnight at 16 °C. After incubation, the microchips can be separated, and the entire mixture (microchips and any remaining tissue fragments) can be deposited into a Falcon tube and incubated with digestion buffer (such as the lysis buffer used in DBiT, or the standard lysis buffer used in bulk tissue dissociation protocols well known to those skilled in the art, such as those used in bulk RNA-seq extraction protocols). The lysate can then be collected, filtered, and subjected to the standard DBiT-seq amplification and NGS library preparation protocol, perhaps with some adjustments to allow for a larger volume of lysate to be collected in this manner. If the tissue adheres to the microchip and is difficult to separate, it can be disrupted mechanically or ultrasonically, such as by placing the mixture in a container within an ultrasonic bath.

[0141] Strategies for Docking Small Chips with Target Substrates with Single or Multiple Regions of Interest

[0142] In some embodiments, multiple microchips will dock with the target substrate, creating multiple active regions, each targeting a different region of interest (ROI) on the target substrate. For example, Figure 12 Shows a glass slide on which multiple tissue sections are mounted, which may be in any position. Thus, each ROI can be located anywhere on the glass slide (possibly within the allowed area, excluding the edges of the glass slide). In this application, each microchip needs to be mechanically pressed onto the glass slide in any position and orientation (rotated around an axis perpendicular to the surface of the glass slide).

[0143] The first strategy to implement a flexible clamping mechanism to accommodate non-uniform tissue spacing is to perform the following steps: (1) Generate a stitched micrograph of a 25×75 mm glass slide carrying tissue sections; (2) Print the image at a 1:1 ratio, e.g., using an inkjet printer; (3) Place a 25×75×3 mm cast acrylic or polycarbonate plate on the printout; (4) Place the non-contact (blank) side of the small chip on top of the plate and aligned with the printed image of the first region of interest, such that the contact (barcoded) side of the small chip will be aligned with the desired ROI after inversion of the transparent substrate; (5) Repeat step 4 for each desired ROI; (6) Peel off the protective laminate from the contact surface of each small chip; (7) Invert the entire assembly and place it on the actual glass slide such that each aligned small chip docks with the glass slide in the desired ROI; (8) Press down on the solid backing to achieve the desired sealing pressure (usually 3 to 30 PSI) for each small chip. If there are 8 small chips and the docking area for each small chip is about half a square inch, a total uniform compressive force of 12 to 120 pounds is required, which can be achieved by a variety of methods familiar to those skilled in the art of mechanical fixation and clamping.

[0144] The second strategy to implement a flexible clamping mechanism to accommodate non-uniform tissue spacing is: during the mixing process, incorporate ferromagnetic nanoparticles into the two-component resin before degassing and curing the microfluidic chip. Since PDMS itself is magnetic, it is possible to compress it onto the target substrate by placing a magnetic element beneath each desired ROI and then placing a magnetic small chip above each ROI.

[0145] Alternative to Small Chips: Thin Strips of Barcoded Gel on Solid Substrates

[0146] Generally speaking, the number of combinations of substrate surface coatings and carrier gels is very large, and a combination that can produce the desired behavior (the gel either remains in the gel carrier or on the solid substrate) can be selected. In the above text, we envisioned that the barcoded gel remains in the gel carrier. However, in view of the above hierarchical discussion, one might wonder whether an equally reasonable strategy might be to deposit the barcoded gel on the solid substrate using a microfluidic chip, with the aim of keeping the gel on the substrate rather than inside the microfluidic chip.

[0147] This method achieves Figure 17A and Figure 17BTwo additional embodiments as shown. These embodiments may be preferred in cases where the selected gel carrier material and the microfluidic device material do not adhere well to each other, making it difficult to retain the barcoded gel within the microfluidic device. In such cases, a substrate can be selected to retain rather than repel the carrier gel. For example, poly-L-lysine-coated slides are known to electrostatically bind negatively charged polymers such as DNA at pH 7.4. Thus, it is also known that negatively charged carrier gels may also be retained by PLL-coated slides at the selected pH. This method has been tested in our laboratory and has been considered successful. However, it is advantageous to retain the gel within the microfluidic chip in cases where the walls of the chip selectively reduce the pore size between channels. Thus, less lateral diffusion is observed when the barcode is retained within the chip rather than on a flat substrate.

[0148] Improving Diffusion of Barcodes from Gel Matrix to Target Substrate by Directed Diffusion

[0149] As previously discussed, chip-based processing delivers fewer active ingredients (e.g., fewer oligonucleotides) than standard flow delivery. While this is mostly due to the small volume of carrier gel in contact with the active area of the target substrate (compared to the volume of the aqueous solution in standard flow), some may also be due to the active ingredients being trapped within the gel matrix, especially if the gel has a relatively small pore size relative to the active ingredient. For example, the size of antibodies may be similar to the pore size of some gels, which may hinder their diffusion from various parts of the carrier gel, especially those furthest perpendicular from the target substrate.

[0150] In such cases, directed diffusion can be employed to transport the active ingredient from the carrier gel to the target substrate. Some examples of directed diffusion include electrophoresis, chemical gradients, and centrifugation.

[0151] An electric field applied to a conductive gel can establish a directed diffusion gradient. For example, in gel electrophoresis, the negative charge of DNA (at typical pH in steady state, i.e., pH 7.4) can promote directed diffusion towards the anode of the electric field generator. For the time being, we only consider those applications of the methods and devices described herein where the active ingredient has a negative or positive charge, or one or more components of the active ingredient have a negative or positive charge, or one or more added elements of the active ingredient have or can be induced (such as one or more charged molecules, or one or more molecules having an induced charge within certain pH ranges) to have a negative or positive charge.

[0152] For example, consider a carrier gel at pH 7.4 tasked with delivering single-stranded DNA (ssDNA) oligonucleotides to a target substrate (e.g., a tissue section). Now consider fixing the tissue section on an indium tin oxide (ITO) and poly-D-lysine-coated glass slide. Due to the ITO component of the surface coating, the glass slide is conductive. Meanwhile, the gel carrier is made of PDMS and has an embedded electrode network to help spread the electric field evenly over the area above the gel strip inside the carrier.

[0153] Since ssDNA develops a negative charge at this pH, placing the cathode in contact with the electrodes embedded in the PDMS structure of the gel carrier and the anode in contact with the ITO-coated conductive glass slide beneath the tissue will drive the oligonucleotide barcodes out of the gel and into the tissue.

[0154] Figure 18 The application of electrophoresis for delivering an active ingredient from a gel carrier into a target substrate is shown. Figure 18 The left and right parts depict imprinting performed with Chip A and Chip B, respectively.

[0155] Exemplary Assays (Other than DBiT-seq)

[0156] Chip-based processing can also be used to support other types of assays besides DBiT, including those aimed at multiplexing tissue treatment conditions. Such methods and systems are described in the context of flow-based processing in US17 / 960,007 entitled "SYSTEM AND METHODS FOR HIGHTHROUGHPUT SCREENING OF TISSUE PREPARATION CONDITIONS", a non-provisional application filed by AtlasXomics inventors (including some of the present inventors). In the chip-based processing described in that application, all protocol steps remain the same except that the flow steps performed using a microfluidic device are replaced with imprinting steps using chips as described herein.

[0157] For example, antibody titration is described in the disclosure, which teaches how to treat a target substrate (tissue section) with different concentrations of an antibody by flowing different concentrations of the antibody sequentially through some or all of the lanes of a 50-channel microfluidic device. In a microchip-based process for such an assay, a microchip can be fabricated by loading gelatin or some other carrier gel with the antibody into the chip, flowing the gel forward into the active area and fixing it in place, peeling the microchip off the flowing substrate, and then pressing it onto the target substrate (i.e., the tissue section). In other words, the active ingredient can be one or more of any number of reagents designed to react with the target substrate, including oligonucleotides, antibodies, enzymes, acids or bases, chromogenic substances (such as peroxidase or other enzymatic staining agents). Depending on the assay target supported by the microchip imprinting process, all active ingredients can have the same concentration or different concentrations.

[0158] By controlling the time at which the active ingredient is released from the gel carrier into the target substrate, time can also be used as a process variable. For example, a gel made of a polymer containing a photocleavable crosslinker reverts to a solution under ultraviolet radiation illumination, thus rapidly releasing the active ingredient previously trapped in the gel matrix. By selectively illuminating sub-regions of the microchip gel array with a suitably arranged ultraviolet illuminator, the experimenter can freely expose different regions of the target substrate to time-varying concentrations of the active ingredient in a way that is difficult or impossible with standard flow devices.

[0159] A tissue section having two or more different cellular microenvironments can benefit from the varying time as a process variable. These two different regions can include the cerebral cortex and the hippocampus, the renal medulla and the cortex, or any tissue section having different regions with different optimal permeabilization conditions. These regions are clearly visible during DBiT operation as regions with high and low fragment counts, respectively.

[0160] In one example, we wish to permeabilize three sub-regions of a tissue, namely N1, N2, and N3, with different permeabilization times. This approach may be particularly applicable when N1 contains some fibrous tissue and is difficult to access, N2 is slightly more accessible, and N3 contains tissue with sparse extracellular structures and is the most accessible. To equalize the accessibility of the reagent between these different microenvironments, we wish to perform permeabilization for T1 = 15 minutes on the first microenvironment N1, T2 = 10 minutes on the second microenvironment N2, and T3 = 5 minutes on N3. The following procedure can be used to achieve these permeabilization times: (1) Prepare a gel in the monomer + crosslinker format, where the crosslinker is photo-cleavable (e.g., by ultraviolet light above a specific intensity threshold). (2) Prepare a liquid of this gel loaded with a permeabilizing agent (e.g., 5% or more gelatin dissolved in water, where 1% of the water is Triton X-100. After crosslinking, the active ingredient will slowly diffuse out of the gel, so the tissue section will not be overly permeabilized before the gel above it is irradiated. When photo-decrosslinked by ultraviolet light, the active ingredient will be rapidly delivered to the tissue section). (3) Mold the loaded gel into a square, approximately the size of the tissue section. (4) Using a photomask + collimated ultraviolet light source or a digital micromirror device (DMD), irradiate only the region corresponding to microenvironment N1 at time T = 0. The DMD has a spatial resolution of less than 1 µm at ultraviolet frequencies. The photomask + collimated light source will likely have a poorer resolution (since it will not be in direct contact with the sample to be irradiated, but will be separated by about a millimeter, causing the light to refract around the edges). (5) After 5 minutes, irradiate only microenvironment N2. (6) After 5 minutes, irradiate only microenvironment N3. (7) Wait for 5 minutes, then wash the entire assembly in warm (37 °C) NEB buffer. The user can choose the shape of the mask or the pattern irradiated by the DMD based on the imaging of the sample or adjacent samples).

[0161] Figures 19A to 19C Relative cycle time data for DBiT operations using monolayer microfluidic chip-based flow processing, bilayer microfluidic chip-based flow processing, and chiplet-based DBiT processing are shown.

[0162] Figures 20A to 20B Electropherograms of multiple different chiplet-based DBiT runs are shown. Figures 21A to 21C Exemplary sequencing datasets generated using chiplet-based processing are shown.

[0163] Figures 22A to 22GShows a comparison of a chip-based run and a standard DBiT-ATAC-seq run performed on tissue sections from the same sample block. In these figures, the term FlowGel refers to chip-based processing. Compares the NGS ATAC library QC and post-sequencing metrics of the chip-based group with the "NoGel" group. Tests comparing group means show that gel-encoded imprinting has no adverse effect on QC metrics and NGS output compared to the standard workflow. Analysis excluded an aberrant chip-based run of DBiT 851 that received an extremely high sequencing depth and looked excellent in the raw sequencing metrics, although its spatial clustering data was poor. This may have been due to incorrect stratification that should have been excluded by visual quality control; however, as an early proof of concept, it was still considered worth trying. Analysis combined 10um and 25um spatial runs. Points: individual DBiT run values. Red lines: box-and-whisker plots showing quartiles, median, and expected variance (1.5 * interquartile range, or minimum and maximum values without outliers beyond the expected variance). Green lines: group means. Black line: overall mean of all plotted values. The X-axis size is proportional to the number of runs within the groups participating in the analysis: post-sequencing metrics: FlowGel n = 6, standard flow n = 18; ATAC NGS library QC metrics: FlowGel n = 18, standard flow n = 40. Mean comparison: Student's two-tailed t-test assuming unequal variances.

[0164] Figure 23 Shows a comparison of chip-based (FlowGel) and spatially resolved ATAC-DBiT-seq based on the fluid flowing through the tissue.

[0165] Figure 24 Demonstrates the use of these chips for co-analyzing spatial whole transcriptome and spatial ATAC on the same tissue section.

[0166] Figure 25 Demonstrates that these chips can be used on substrates different from tissue sections. Specifically, these are spatially resolved ATAC DBiT of cells printed on glass slides.

[0167] List of Terms

[0168] Hereinafter, we refer to existing composition printing methods as "flow-based" methods to distinguish them from the "chip-based" methods described herein. This imprinting method forms an array of gel strips containing the composition inside the microfluidic chip by flowing the composition over a blank substrate, and then contacts the array with the substrate for a long enough time for the composition in the gel to interact with the substrate. These methods require the following key components, along with exemplary but non-limiting embodiments:

[0169] 1. Active ingredient: The material to be deposited on the target substrate in a defined spatial pattern

[0170] 2. Active ingredient array: Parallel features filled with active ingredients, enabling the assays supported by the method. Examples: DBiT-seq, T-Rec.

[0171] 3. End user - An individual or group that imprints the active ingredient array onto the target substrate.

[0172] 4. Gel carrier / microchip (possibly a trade name) - A device that contains a gel with the ingredients and delivers the consumable to the end user.

[0173] a. A preferred embodiment is a microfluidic chip

[0174] i. AtlasXomics Suburbia A / B chip, with resolutions of 5μm, 10μm, 25μm, 50μm

[0175] ii. AtlasXomics Portal A / B chip, with a resolution of 25μm

[0176] iii. FlowGel SuperChip, with resolutions of 10μm, 15μm, 25μm and 50μm

[0177] iv. Wafer-sized master chip

[0178] b. The role of the gel carrier is to deliver the active ingredient to the target substrate.

[0179] c. In some preferred embodiments, the pattern consists of dozens or hundreds of parallel thin lines, with widths and spacings of 25um or less.

[0180] d. In other embodiments, the pattern consists of several or dozens of so-called "millifluidic" chambers, covering a larger area of the target substrate (e.g., 1×1mm, or 1×2mm, or similar shapes and sizes).

[0181] 5. Flow-through substrate

[0182] a. A flow-through substrate such as made of glass, with or without a hydrophobic coating, such as commercially available products like RainX TM

[0183] b. Or a flow-through substrate made of silicon, with or without a hydrophobic coating, such as commercially available products like RainX TM

[0184] c. A flow-through substrate made of Teflon, PEEK, or other hydrophobic polymers that cannot adsorb or absorb the carrier gel, or any of a variety of other highly non-adsorbent polymer substrates

[0185] d. Alternatively, in a preferred embodiment, a flow-through substrate made of a flexible plastic sheet, such as polyethylene (PET) or polycarbonate (PC) with a thickness of 100 μm, 200 μm, or less than 100 μm. A key advantage of the flexible sheet is that it can be peeled from the gel carrier without bending the gel carrier itself, which allows the carrier gel to be better retained within the gel carrier without having to peel the gel carrier from the flow-through substrate (which would have to be done if the flow-through substrate were rigid, including a glass slide or other rigid material).

[0186] 6. The target substrate, after the gel carrier is delaminated from the blank flow-through substrate, to which the active ingredient is delivered by imprinting. For DBiT-seq, the target substrate is a thin (10 μm or thinner) fixed tissue section on a coated glass slide. In other cases, the target substrate can be a thicker tissue sample (e.g., a tissue sample 10 μm or thicker, up to 100 μm thick cut on a vibratome), a tissue block (e.g., a tissue block recovered from a tissue biopsy), printed cells, organoids, plants, absorbent materials for paints or other inks, the outer layer of an animal (e.g., the skin of a human arm).

[0187] 7. The carrier gel material impregnated with the active ingredient, which flows as a liquid over the flow-through substrate on a microfluidic chip, is embedded in place within the microfluidic chip (preferably by changing the temperature or other methods that do not rely on adding an external catalyst, since the gel is inaccessible before delamination), and is delaminated from the flow-through substrate, where enough gel is retained by the chip (rather than the substrate) to support subsequent application of the impregnated reagent to the target substrate and delivery into the target substrate. The potential compatible materials for making the carrier gel material are listed here:

[0188] a. Water-absorbing gels (“hydrogels”). As in “a three-dimensional cross-linked polymer system capable of absorbing a large amount of water or biological fluid between its polymer chains to form a water-containing semi-solid / solid gel network”

[0189] i. Physical / reversible hydrogels

[0190] 1. Thermally controlled

[0191] a. Gelatin

[0192] b. Pectin

[0193] c. Poloxamer

[0194] d. PL(G)A-based triblock gelators

[0195] e. Poly(N-isopropylacrylamide) (PNIPAM)-poly(phosphorylcholine)-PNIPAM

[0196] f. Asymmetric triblock copolymer formed by PEG, PLA and poly(L-glutamic acid)

[0197] g. Chitosan solution and glycerol-2-phosphate

[0198] h. Hydroxypropyl cellulose

[0199] i. Thermosensitive methyl cellulose, thermogel of alginate aqueous solution blended with various salts such as CaCl2, Na2HPO4 and NaCl

[0200] 2. Ion binding

[0201] a. Mixture of quaternized chitosan (N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC)) and glycerol phosphate (GP)

[0202] b. Anion (methacrylic acid (MAA)) and cation (dimethylaminoethyl methacrylate (DMAEMA)) polymer-coated dextran microspheres derived from 2-hydroxyethyl methacrylate

[0203] 3. Hydrogen bond mediated

[0204] a. PVA

[0205] b. PVA combined with other polymers such as chitosan ("cryogel")

[0206] i. PVA cryogel containing minocycline and gentamicin

[0207] c. Blend of two or more natural polymers such as hyaluronic acid-methyl cellulose, gelatin-agar and starch-carboxymethyl cellulose

[0208] d. Composite cryogel of metronidazole with carboxymethyl tamarind kernel polysaccharide and PVA

[0209] 4. Gels based on stereocomplexation

[0210] a. Poly(lactide) blocks with L stereochemistry and D stereochemistry

[0211] b. Isotactic and syndiotactic poly(methyl methacrylate) (PMMA)

[0212] 5. Gels based on supramolecular chemistry

[0213] a. Reversible hydrogel complex of PEO polymer and α-cyclodextrin

[0214] b. The dextran grafted with PPO forms a hydrogel with β-cyclodextrin

[0215] c. The PEO-poly(R-3-hydroxybutyrate) (PHB)-PEO triblock copolymer complexes with α-cyclodextrin to form a self-assembled hydrogel network

[0216] ii. Chemical / permanent gels that deliver active ingredients by diffusing into the target substrate rather than through gel dissociation.

[0217] 1. Crosslinked gels

[0218] a. Dialdehydes (such as glyoxal, especially glutaraldehyde 46) form covalent imine bonds with the amino groups of chitosan through the Schiff reaction

[0219] b. Dextran-tyramine and hyaluronic acid-tyramine covalently bound using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) as crosslinkers

[0220] c. Genipin crosslinks chitosan or gelatin, or molecules containing amino-capped groups such as PEG, N,O-carboxymethyl chitosan, and BSA

[0221] 2. Polymer-polymer crosslinking or hybrid polymer network (HPN)

[0222] a. Crosslinking of vinyl sulfone-functionalized dextran with thiolated PEG

[0223] 3. Photo-crosslinking

[0224] a. Azide groups (-N3) incorporated into the chitosan polymer chain

[0225] b. Thermosensitive chitosan-Pluronic hydrogel in which both polymers are functionalized with photosensitive acrylate groups (CH2=CHCOO-) by ultraviolet light irradiation

[0226] c. Modifying chitosan with photo-reactive azidobenzoic acid and PEG with arginylglycylaspartic acid peptide

[0227] 4. Enzyme crosslinking

[0228] a. Gelatin hydrogel crosslinked by microbial TG (mTG)

[0229] b. HRP-catalyzed injectable tyramine-modified hyaluronic acid (HA-Tyr)

[0230] c. Developing an enzyme-crosslinked injectable hydrogel from chitosan derivatives, chitosan-glycolic acid, and phloretic acid using HRP and H2O2

[0231] 5. Interpenetrating network (IPN)

[0232] The foregoing description of the various embodiments is for illustrative and descriptive purposes only. They are not intended to be exhaustive or to limit the system to the disclosed forms. Accordingly, many modifications and variations will be obvious to practitioners skilled in the art. Additionally, the foregoing disclosure is not intended to limit the system.

Claims

1. A method, comprising: Directly fixing a flowing substrate to a microfluidic chip including a plurality of channels; Flowing a first reagent embedded in a first gel carrier material through a first channel among the plurality of channels and flowing a second reagent embedded in a second gel carrier material through a second channel among the plurality of channels; Adjusting one or more material properties of the first gel carrier material and the second gel carrier material disposed within the plurality of channels to restrict further movement of the first reagent and the second reagent within the microfluidic chip and form a microchip.

2. The method according to claim 1, further comprising: Separating the flowing substrate from the microchip.

3. The method according to claim 1, further comprising: Cutting the microchip into a plurality of pieces to produce one or more microchips including at least a first microchip, wherein the first microchip includes a first portion of the first channel and a second portion of the second channel, and wherein the first portion and the second portion of the first channel and the second channel included in the first microchip extend from a first end of the first microchip to a second end of the first microchip.

4. The method according to claim 3, further comprising: Inspecting the one or more microchips for defects; And Discarding any microchip determined to be defective among the one or more microchips during the inspection.

5. The method according to claim 3, wherein the first channel is parallel to the second channel, and the first channel and the second channel extend linearly from the first end of the first microchip to the second end of the first microchip.

6. The method according to claim 3, wherein cutting the microchip into a plurality of pieces produces a second microchip and the first microchip.

7. The method according to claim 3, further comprising: Fixing the first microchip to a substrate of interest; And Directing the first reagent and the second reagent to at least a first isolation region and a second isolation region of the substrate of interest.

8. The method according to claim 1, wherein adjusting the one or more material properties of the first gel carrier material and the second gel carrier material includes reducing the temperature of the first gel carrier material and the second gel carrier material to increase the viscosity of the first gel carrier material and the second gel carrier material.

9. The method according to claim 1, further comprising: Fixing the microchip to a substrate of interest; And Directing the first reagent and the second reagent to at least a first isolation region and a second isolation region of the substrate of interest.

10. A method, comprising: Applying a microchip to a substrate of interest, wherein the microchip includes a plurality of parallel channels extending from a first end of the microchip to a second end of the microchip opposite the first end, the plurality of channels including a first channel and a second channel, the first channel being filled with a first reagent embedded in a first gel carrier material, and the second channel being filled with a second reagent embedded in a second gel carrier material; Fix the microchip to the substrate of interest; Increase the temperature of the microchip to allow the first reagent and the second reagent to flow onto the substrate of interest and interact with the substrate of interest; And Remove the microchip from the substrate of interest after a predetermined incubation time.

11. The method according to claim 10, wherein applying the microchip to the substrate of interest includes applying a plurality of microchips to different regions of the substrate of interest.

12. A microchip, comprising: A first substrate defining a plurality of channels, wherein the plurality of channels extend from a first end of the first substrate to a second end of the first substrate opposite the first end; A first reagent embedded in a gel carrier material filling a first channel of the plurality of channels; A second reagent embedded in the gel carrier material filling a second channel of the plurality of channels; And A second substrate covering the plurality of channels.

13. The microchip according to claim 12, wherein the temperature of the microchip increases the viscosity of the gel carrier material to above a threshold, thereby restricting the movement of the first reagent and the second reagent within the first channel and the second channel.

14. The microchip according to claim 12, wherein the gel carrier material is lyophilized to restrict the movement of the first reagent and the second reagent within the first channel and the second channel.

15. The microchip according to claim 12, wherein the second substrate covering the plurality of channels is removed before imprinting onto a substrate of interest.

16. The microchip according to claim 15, wherein after the microchip is fixed to the substrate of interest, the gel matrix is reversely crosslinked by heat to release the active reagent.

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