WO2026024999A1 - Controlled evaporation and density equalization of hydrogel microbeads - Google Patents

Abstract

Hydrogel microbeads are reduced in size by a method comprising controlled evaporation of solvent from HMBs through an oil carrier phase, wherein the shrinking hydrogel increases in density until it reaches a density equal to the carrier phase and becomes neutrally buoyant, at which point HMBs sink and no longer change significantly in size, wherein the evaporation continues until all hydrogels reach uniform size, wherein the volume reduction is controllable by modulating the oil and aqueous phases. Methods, materials and systems for selective magnetic separation of cells or molecules in microcompartments, include loading a mixture of target and nontarget material into the microcompartments, selectively labeling the target material with a magnetic particle, and magnetically separating the target material from the non-target material to isolate and recover the target material.

Description

Controlled Evaporation and Density Equalization of Hydrogel Microbeads

[001] Introduction

[002] Hydrogel microbeads (HMBs) contain networks of polymers that can absorb and retain large amounts of solvents. They are typically 1-50 microns in diameter. HMBs have numerous biomedical and environmental applications in drug delivery, tissue engineering, environmental remediation, diagnostics, and sequencing. HMBs can be synthesized from polymers and modified with protein, nucleic acid, and chemical moieties to endow them with unique properties including responsiveness to environmental stimuli such as temperature and pH changes. Tailored modifications allow for controlled and targeted drug delivery in personalized medicine, biocompatibility for in vivo applications, or biodegradability for environmental use. [003] The versatility of HMBs extends to their capacity to support cell viability and function, making them important scaffolds in tissue engineering and regenerative medicine. They provide an ideal scaffold for cell growth and differentiation, crucial in developing artificial tissues and organs. Tn cosmetic and personal care products, the high-water content has enhanced moisturizing and texturizing properties, improving product efficacy and consumer experience. The agriculture and environmental sectors utilize HMBs to absorb and release moisture and nutrients for soil management and capture pollutants in water treatment processes. In research, HMBs are a critical part of many single-cell analysis platforms. Microbeads are used to precisely barcode cellular material for single-cell RNA, ATAC, and proteomic workflows. Single-cell sequencing has become an important tool in all biological research.

[004] The size of HMBs is important for various applications. For instance, in cell therapy and transplantation, HMBs can protect cells and enable efficient nutrient and oxygen exchange, and the bead size is crucial for ensuring cell survival. Beads that are not correctly sized can hinder the provision of an adequate environment for cells, potentially impacting their integration and function post-transplantation. In the field of tissue engineering, HMBs act as 3D scaffolds, promoting cell growth and differentiation. The size of the beads influences tissue structure development, with optimal sizing supporting natural 3D growth and enhancing the structure and function of the engineered tissues. Additionally, in sensors and diagnostic tools, the size determines surface area and reactivity. Appropriately sized beads increase the surface area available for interactions with biomarkers or target molecules, aiding in enhancing the sensitivity and accuracy of sensors. The diversity in microbead applications necessitates methods to effectively tune bead size while maintaining monodispersity. This level of control is vital for advancing HMBs in specialized and demanding applications. [005] Typically, HMBs vary in size from one to several hundred micrometers. The size is a critical determinant of their function but often needs to change during synthesis, use, or analysis. For instance, HMBs used for sequencing applications often range from 50 to 100 m, which is too large for subsequent sorting on commercial Fluorescence Activated Cell Sorting (FACS) systems. Such systems typically require objects for sorting to be smaller than 30 pm.

[006] Magnetic-activated cell sorting (MACS) isolates cells using magnetic nanoparticles attached to antibodies. If the antibody recognizes a protein antigen on the cell’s surface, it binds to the antigen, and the cell becomes magnetic. Cells are washed to remove unbound antibodies, and the labeled cells are brought into contact with a magnet. The magnet retains only antibody- labeled cells, and the rest can be removed by washing. In this way, cells that contain a surface antigen can be isolated for further studies. This technique is widely used in immunology. Using this approach, it is easy to isolate cells based on surface antigens. However, equivalent methods for isolating cells based on non-protein targets are lacking.

[007] Microcompartments are pico-to-nanoliter reaction vessels that can be loaded with cells, biomolecules, and reagents. They include water-in-oil emulsions (droplets), hydrogel microspheres, or thin-shelled capsules. Microcompartments can be made with microfluidic or non-microfluidic methods. They can be used to separate biological/chemical reactions such that each microcompartment has distinct contents that undergo processing independent of other microcompartments. Microcompartments are now widely used in single-cell genomics.

[008] Digital PCR amplifies a target nucleic acid inside microcompartments. If this amplification reaction is coupled to a fluorescent dye (e.g. TaqMan), the starting molecules can be detected and/or sorted by measuring the fluorescence of the microcompartment, providing a basis for digital PCR molecule counting.

[009] Summary of the Invention

[010] The invention provides methods of controlled evaporation and density equalization of hydrogel microbeads.

[011] In an aspect the invention provides a method for uniformly reducing the size of hydrogel microbeads (HMBs), comprising controlled evaporation of solvent from HMBs through an oil carrier phase, wherein the hydrogel shrinks, and the shrinking hydrogel increases in density until it reaches a density equal to the carrier phase and becomes neutrally buoyant, at which point the HMBs sink and no longer change significantly in size, wherein the evaporation continues until the HMBs reach uniform size, wherein the volume reduction is controllable by modulating the oil and aqueous phases (e.g. by adjusting the initial density of the hydrogel solution or by altering the density of the oil). [012] In embodiments the method is substantially as shown in Fig. 1.

[013] In embodiments the method is substantially as shown in Fig. 1 , and comprising controlled evaporation of solvent from HMBs through an oil shell, wherein the hydrogel shrinks, and the shrinking hydrogel increases in density until it reaches a density equal to the oil phase and becomes neutrally buoyant, wherein initially, the beads are placed in a denser-than-water oil phase, such as HFE-7500 with a surfactant or any commercially available oil suitable for droplet generation, the mixture is vortexed with sufficient energy to disperse the beads within the oil, resulting in an emulsion where each droplet encapsulates a single bead, this emulsion is then heated (e.g. to approximately 60 °C for agarose hydrogels), while airflow and oil are continuously supplied during heating to facilitate evaporation and replenish the reduced oil phase, and additionally, the emulsion is intermittently stirred gently, wherein evaporation primarily occurs at the droplets on the top layer — at the interface between oil and air — causing them to diminish in size, wherein this reduction in volume leads to an increase in both the hydrogel concentration and the density of the droplets, wherein intermittent stirring disrupts the settled droplets, ensuring their homogeneous suspension in the oil, wherein the increased density causes the shrunken droplets to reach a density similar to that of the oil, which prevents them from ascending to the air-oil interface to undergo further evaporation, wherein eventually, once all the droplets attain a certain density level, the top emulsion layer dissipates, and the droplets, now with a density matching that of the oil, are uniformly suspended within the oil phase, wherein with the droplets no longer at the air-oil interface, evaporation and subsequent shrinking cease, wherein this process of consistent density equilibrium effectively and uniformly reduces the size of the HMBs by using controlled evaporation and density equalization within the oil phase.

[014] In embodiments, the method is implemented using an apparatus substantially as shown in Fig. 2.

[015] In other embodiments, magnetic labeling is applied to isolate target-containing beads without shrinking. The two processes are modular and may be combined or used separately depending on assay need. The methods provide improvements to both fluorescent activated sorting (FACS) and magnetic sorting. In the case of FACS, the fluid passed a narrow gauge nozzle, and larger beads disrupt the droplet formation, causing splatter and sometimes clog the nozzle. All FACS machines have an upper limit on the size of objects that they can sort. Hence, the reduced size bead of the invention expand the applications of FACS. Magnetic enrichment can also be limited by larger objects, which is related to the Stokes drag, whereas the reducedsized beads of the invention move faster, making them easier to separate. Also, the smaller beads have reduced tendency to clog commercial magnetic columns. [016] In embodiments, the method further comprises FACS-based sorting of the resultant reduced-sized hydrogel microbeads, wherein the bead shrinking may be applied independently of magnetic labeling.

[017] In embodiments, the method further comprises enriching the resultant reduced-sized (shrunken) hydrogel microbeads on a FACS machine, substantially as shown in Fig. 4.

[018] In embodiments, the method further comprises FACS-based sorting of the resultant reduced-sized hydrogel microbeads, and selective isolation of a transcriptome , such as singlecell RNA sequence a rare cell population, substantially as shown in Fig. 5.

[019] In embodiments, the method further comprises deploying the resultant uniformly reduced size hydrogel microbeads (HMBs) for magnetic separation of hydrogels.

[020] In embodiments, the method further comprises deploying the resultant uniformly reduced size hydrogel microbeads (HMBs) in a method for selective magnetic separation of cells or molecules in microcompartments, substantially as shown in Fig. 7, Fig. 8 or Fig. 9.

[021] In an aspect the invention provides a method for selective magnetic separation of cells or molecules in microcompartments, substantially as shown in Fig. 7, Fig. 8 or Fig. 9.

[022] In an aspect the invention provides a method of enriching cells, nucleic acids, proteins, or chemicals using magnetic labeling substantially as shown in Fig. 7, and comprising:

[023] a) providing microcompartments that are sub-pico to nano-liter vessels created using microfluidic devices or other techniques, wherein the microcompartments can be impermeable or semi-permeable, allowing for the selective retention and labeling of their contents, wherein the microcompartments are loaded with a sample containing target and non-target material, such as wherein the target is a nucleic acid and is amplified to increase the signal;

[024] b) linking the target material to a magnetic particle so that the microcompartment with the target material becomes magnetic;

[025] c) washing to remove unbound magnetic particles;

[026] d) purifying using magnets (e.g. column or tube-based purification) to isolate compartments containing targets of interest, and

[027] e) recovering the contents of compartments after purification.

[028] In an aspect the invention provides a method of magnetic-activated nucleic acid cell sorting to isolate cell genomes and transcriptomes based on nucleic acid biomarkers, substantially as shown in Fig. 8, and comprising:

[029] a) providing cells encapsulated within microcompartments and lysed, releasing the genome and transcriptome for biochemical reactions, and adding primers targeting the genome or transcriptome; [030] b) amplifying target nucleic acids using polymerase chain reaction (PCR) or another enzymatic amplification strategy, wherein primers can be modified to enable magnetic labeling, typically using 5 ’ biotin.

[031] c) adding affinity streptavidin labeled magnetic nanoparticles to bind to the biotinylated, or functional equivalent, and washing away unbound streptavidin (or functional equivalent) and magnetic nanoparticles;

[032] d) placing the solution containing magnetic and non-magnetic microcompartments near a magnet to isolate only magnetic micro compartments, wherein non magnetic micro compartments are washed away, and the magnet is removed and the microcompartment’ s shell or hydrogel is digested or removed to recover cell material of interest.

[033] In an aspect the invention provides a method of purification of beads with specific nucleic acid barcodes substantially as shown in Fig. 9, and comprising isolating barcoded beads of interest and, therefore, the single-cell material attached to those beads, wherein cDNA reverse transcribed by barcoded primers on the bead is sequenced, and after bioinformatic analysis, cells of interest are identified based on their transcriptional profiles, wheren the barcodes associated with these cells are used to purify the corresponding beads, which can be effected by hybridization of complementary barcodes containing magnetic nanoparticles, magnetic separation, and deep sequencing of the transcriptome, wherein multi-omic embodiments, only one sequencing modality is needed to identify barcodes of interest, for example, in RNA and protein methods (e.g. CITE-seq) or RNA and gRNA methods (Perturb- seq), barcodes can be identified by the transcriptome, followed by barcode-based enrichment using complementary barcodes containing magnetic nanoparticles, wherein the second modality (protein or sgRNA in this example), from only cells of interest, is then sequenced.

[034] In an aspect the invention provides a method for selective magnetic separation of cells or molecules in microcompartments (flexible picoliter to nanoliter-sized chambers, such as hydrogel beads or capsules), comprising loading a mixture of target and nontarget material into the microcompartments, selectively labeling the target material with a magnetic particle, and magnetically separating the target material from the non-target material to isolate and recover the target material, wherein the method is magnetically separating the microcompartment using the target material, i.e, not just recovering the target, but recovering everything in the microcompartment.

[035] In embodiments the subject methods further comprise:

[036] a) Enrichment of specific nucleic acids from complex samples, e.g. combined with digital PCR to isolate molecules or cells that contain target RNA or DNA sequences; [037] b) Magnetic-activated nucleic acid cell sorting, e.g. isolating cell material based on the amplification of specific nucleic acid sequences, labeling of amplicons with magnetic particles, and the isolation using a magnet of the entire agarose bead containing cell ‘omics (genome, epigenome, proteome, transcriptome);

[038] c) Purification of beads with specific nucleic acid barcodes, e.g. enabling selection of specific chemicals from within DNA-barcoded chemical libraries created inside a compartment, wherein the corresponding DNA tag is used to purify chemicals of interest for magnetic separation;

[039] d) CRISPR screens, e.g. a reporter assay, wherein the reporter can be any amplicon that is generated via digital PCR; and/or

[040] e) Generation of barcoded beads for single-cell genomics applications, e.g. providing large numbers of barcoded beads with high barcode diversity and with no non-barcoded beads, wherein the labeling of compartment material with magnetic particles can occur using a variety of established methods, including protein interactions (e.g. biotin-streptavidin), chemical (e.g. click), nucleic acid hybridization, or non-specific (hydrophobic interactions).

[041] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[042] Brief Description of the Drawings

[043] Fig. 1. Schematic drawing of bead shrinking principle.

[044] Fig. 2. Setup for evaporation.

[045] Fig. 3. Images of beads before and after shrinking

[046] Fig. 4. Enrichment of shrunken beads on a FACS machine. Beads containing intact provirus were identified based on their fluorescence signals and sorted using a FACS machine. The beads were displayed before and after sorting, and the AL594 intensity v. FAM intensity for the initial and post-sorted beads was determined.

[047] Fig. 5. Application of the bead shrinking method to FACS sort and single-cell RNA sequence a rare cell population. The figure also shows the experimental design to validate the selective isolation of transcriptomes from rare intact SIV pro virus -harboring cells. The beads were stained with SYBR™ Green I and sorted via FACS. The sorting of triple-positive beads were sorted, and PCR was conducted, and products were purified, followed by size validation. [048] Fig. 6. Application of bead shrinking to purify beads with specific cellular material magnetically. Fig. 6 shows agarose beads attached with a DNA molecule containing a 3 ’-biotin, mixed with agarose beads without attached DNA or biotin. Fig. 6 also shows that the biotin was labelled with streptavidin magnetic nanoparticles. Fig. 6 also shows the purification of agarose- DNA-biotin-Streptavidin-nanoparticle complexes using a magnet. Fig. 6 also shows the fluorescence analysis of the negative and positive fractions after magnetic purification by flow cytometer.

[049] Fig. 7. Schematic drawing of principle. Method for enriching cells, nucleic acids, proteins, or chemicals using magnetic labeling. 1) Microcompartments are sub-pico to nano-liter vessels created using microfluidic devices or other techniques. 2) Microcompartments can be impermeable or semi-permeable, allowing for the selective retention and labeling of their contents. 3) Microcompartments are loaded with a sample containing target and non-target material. In some applications, the target is a nucleic acid and is amplified to increase the signal. 4) The target material is linked to a magnetic particle so that the microcompartment with the target material becomes magnetic. 5) Washing is used to remove unbound magnetic particles. 6) Column or tube-based purification with magnets is used to isolate compartments containing targets of interest. 7) Contents of compartments are recovered after purification.

[050] Fig. 8. Magnetic-activated nucleic acid cell sorting. Application of the technique to isolate cell genomes and transcriptomes based on nucleic acid biomarkers. 1) Cells are encapsulated within microcompartments and lysed, releasing the genome and transcriptome for biochemical reactions. Primers targeting the genome or transcriptome are added. 2) Target nucleic acids are amplified using polymerase chain reaction (PCR) or another enzymatic amplification strategy. Primers can be modified to enable magnetic labeling, for example using a 5’ biotin. 3) Streptavidin magnetic nanoparticles are added and bind to the biotinylated sequences. Unbound streptavidin and magnetic nanoparticles are washed away. 4) The solution containing magnetic and non-magnetic microcompartments are placed on a magnet to isolate only magnetic micro compartments. Non magnetic micro compartments are washed away. The magnet is removed and the microcompartment’s shell or hydrogel is digested or removed to recover cell material of interest.

[051] Fig 9. Purification of beads with specific nucleic acid barcodes. Barcoded beads are used routinely in single-cell RNA sequencing, DNA sequencing, protein sequencing, and chromatin sequencing applications. This technology can isolate barcoded beads of interest and, therefore, the single-cell material attached to those beads. In this embodiment, cDNA reverse transcribed by barcoded primers on the bead is sequenced. After bioinformatic analysis, cells of interest are identified based on their transcriptional profiles. The barcodes associated with these cells are used to purify the corresponding beads. This can be completed by hybridization of complementary barcodes containing magnetic nanoparticles, magnetic separation, and deep sequencing of the transcriptome. In multi-omic studies, only one sequencing modality is needed to identify barcodes of interest. For example, in RNA and protein methods (e.g. CITE-seq) or RNA and gRNA methods (Perturb-seq), barcodes can be identified by the transcriptome, followed by barcode -based enrichment using complementary barcodes containing magnetic nanoparticles. The second modality (protein or sgRNA in this example), from only cells of interest, is then sequenced.

[052] Description of Particular Embodiments of the Invention

[053] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes. [054] I. Controlled Evaporation and Density Equalization of Hydrogel Microbeads [055] Herein, we demonstrate a method for reducing the size of HMBs without increasing their size distribution. A uniform reduction in HMB size is achieved through the controlled evaporation of solvent from HMBs through an oil shell. The shrinking hydrogel increases in density until it reaches a density equal to the oil phase and becomes neutrally buoyant. Initially, the beads are placed in a denser-than-water oil phase, such as HFE-7500 with a surfactant or any commercially available oil suitable for droplet generation. The mixture is vortexed with sufficient energy to disperse the beads within the oil, resulting in an emulsion where each droplet encapsulates a single bead. This emulsion is then heated (to approximately 60 °C for agarose hydrogels), while airflow and oil are continuously supplied during heating to facilitate evaporation and replenish the reduced oil phase. Additionally, the emulsion is intermittently stirred gently.

[056] Evaporation primarily occurs at the droplets on the top layer — at the interface between oil and air — causing them to diminish in size. This reduction in volume leads to an increase in both the hydrogel concentration and the density of the droplets. Meanwhile, intermittent stirring disrupts the settled droplets, ensuring their homogeneous suspension in the oil. With the iteration of this, the increased density causes the shrunken droplets to reach a density similar to that of the oil, which prevents them from ascending to the air-oil interface to undergo further evaporation. Eventually, once all the droplets attain a certain density level, the top emulsion layer dissipates, and the droplets, now with a density matching that of the oil, are uniformly suspended within the oil phase. With the droplets no longer at the air-oil interface, evaporation and subsequent shrinking cease. This process of consistent density equilibrium effectively and uniformly reduces the size of the HMBs by using controlled evaporation and density equalization within the oil phase. The principle of this size-reduction technique is illustrated in Fig. 1, and the apparatus used in the procedure is depicted in Fig. 2.

[057] The final size of the reduced hydrogel beads can be manipulated by adjusting the initial density of the hydrogel solution or by altering the density of the oil. According to Stokes' law, the velocity at which particles rise to the surface is determined by the density difference between the particles and the solvent. The equation suggests that if additives like sucrose or glycerol are included in the hydrogel solution, the density of the droplets increases during the evaporation process.

[058] V= (2 g r² Ap)/9 q, where: V is the distance traveled by the particle; Zip is the density difference; g is the gravity; r] is the viscosity of solvent; and r is the size of particle.

[059] Consequently, the droplets quickly reach a density of oil, preventing them from rising further. By varying the concentration of additives or the density of oil, the final size of the beads can be controlled. This control is essential for applications where precise bead size changes are critical to the functionality and application outcomes.

[060] The invention can be used in any field where HMBs are applied to change size, provided the gel allows for evaporation. In embodiments the process is reversible. In embodiments the invention is utilized in FACS (<20 pm). Practical pplications include: [061] Nucleic acid cytometry. We and others have developed workflows for detecting nucleic acids in single cells. These workflows rely on water-in-oil assays to retain the molecular signal. One application of bead shrinking leverages a FACS machine, instead of a droplet sorter, to sort these cells. Cells of interest can be detected using single-cell digital analysis (PCR, LAMP, other), processed using workflows similar to FIND-seq (or another), followed by shrinking of HMBs and FACS sorting.

[062] Tissue engineering. In some cases, it may be useful to encapsulate cells, or groups of cells in a hydrogel scaffold. The size/scaffold properties of HMBs during encapsulation may need to differ from the size/properties for tissue formation/printing. This technique can be used to change the size or elasticity of the gel for this purpose.

[063] Barcoded beads generation. Digital PCR on single molecules inside droplets can amplify a single template. If a random barcode with PCR handles is used with HMBs, barcoded beads can be created for single-cell sequencing applications. To ensure that a single template is present, most droplets are empty. This means that droplet PCR is an inefficient method for making barcoded beads because most beads have no barcode. Shrinking, followed by enrichment for barcoded beads by FACS can overcome this limitation. [064] Particle or molecule capture. Shrinking can change the pore size of the hydrogel, allowing HMBs to trap particles, cells, polymers, nucleic acids, or other objects within the gel. This may include the trapping of magnetic nanoparticles to create multi-hydrogel beads for advanced molecular or magnetic imaging techniques.

[065] Triggering of reactions. HMB shrinking can increase the concentration of a target analyte and therefore facilitate its detection or trigger a chemical or biological reaction.

[066] Drug or gene delivery. HMB shrinking can facilitate the delivery of drug or gene cargo and may be used to modulate biocompatibility or release kinetics, or change cell type (or organ) specific targeting and bioavailability.

[067] Examples: Method for Uniform Size Reduction of Hydrogel Microbeads via Controlled Evaporation and Density Equalization

[068] Agarose beads shrinking. Agarose beads, made from 2% agarose (~55 pm), were successfully reduced to ~20 pm in size (see Fig. 3). To achieve this, 0.5 mL of the agarose beads were resuspended in PBS and then emulsified in 3 mL of QX200™ Droplet Generation Oil for EvaGreen (Bio-Rad, catalog no. 1864005) using Particle-templated Instant Partition (PIP) emulsification. The emulsion was heated to 60 °C with an air flow of 1 psi applied. During the bead shrinking process, the oil was supplied at a consistent rate of 7 mL/hr. The mixture was intermittently stirred — 30 s of stirring followed by a 10 min interval — for an hour to ensure even shrinking. After the procedure, the shrunken beads suspended in oil were cooled at 4 °C for one hour, allowing the agarose beads to solidify. For further processing, 0.25 mL of acetone was added to 0.5 mL of the bead-oil mixture, and the solution was thoroughly vortexed. The beads were then pelleted by centrifugation for 3 minutes at 3000 x g and 4 °C, and the supernatant was discarded. The resulting bead pellet was resuspended in a mixture of 0.5 mL HFE-7500 and 0.25 mL acetone, centrifuged again, and finally resuspended in PBS-T with 0.1% Tween- 20.

[069] Intact SIV provirus-harboring genome/transcriptome enrichment. Agarose beads encapsulating the genome and transcriptome of intact SIV provirus-harboring cells were prepared as previously described with the following modifications. Initially, the allyl-modified agarose backbone was functionalized with acrydite-modified forward primers specific to the SIV pol/env/LTR regions for the TaqMan assay, along with Oligo d(T) primers for mRNA capture. The concentration of the functionalized pol/env/LTR primers was adjusted to 1 pM, and the Oligo d(T) primers were adjusted to 8 pM using a standard 2% agarose solution. SIV1C cells, a cell line harboring intact SIV provirus, were encapsulated within the droplets at a ratio of 1 cell per 10 droplets, along with the functionalized agarose and a lysis buffer containing Proteinase K and LiDS. Following complete lysis, the provirus-capturing agarose beads, approximately 55 pm in size, were transferred to the aqueous phase by breaking the droplets with 20% PFO in HFE-7500. The mRNA released during lysis was hybridized to the Oligo d(T) via poly(A) tail hybridization. Given the large size of the genome, which prevents diffusion out of the agarose beads, some of beads (-10%) contained a provirus. Reverse transcription was then performed using a master mix containing TSO (10 pM), dNTPs (1 mM), Betaine (1 M), PEG (8 kDa, 7.5% w/v), Maxima H Minus Reverse Transcriptase (2 U/pL), and NxGen RNase Inhibitor (0.5 U/pL). Following reverse transcription, the beads containing cDNA were thoroughly washed in 0.1% (v/v) Tween-20 solution. These beads were then re-injected into droplets containing the pol/env/LTR TaqMan assay components, including reverse primers for pol/env/LTR detection (0.9 pM each), TaqMan probes for pol/env/LTR detection (0.25 pM each), and TaqPath™ ProAmp™ Master Mix. The emulsion underwent thermocycling to generate TaqMan signals and to tether the amplicons onto the agarose beads. After thermocycling, the beads were again collected into the aqueous phase. They were treated with 0.15 M NaOH to denature the tethered amplicons into single strands, and then hybridized with complementary oligonucleotides (hyb probes) tagged with FAM and Cy3 for pol and env, respectively. Following the staining process, the beads underwent a shrinking procedure. Beads containing intact provirus (dual-positive for both pol and env signals) were identified based on their fluorescence signals and sorted using a FACS machine, as illustrated in Fig. 4. Fig. 4 also shows the beads before and after sorting, respectively, highlighting the sorting process's efficacy in enriching dual-positive beads from 12.6% to 96.0%. Fig. 4 also shows the AL594 intensity v. FAM intensity for the initial and postsorted beads.

[070] Fig. 5 shows the experimental design to validate the selective isolation of transcriptomes from rare intact SIV pro virus-harboring cells. SIV1C cells were mixed with N1H/3T3 cells (non-infected) at a 1: 100 ratio. After encapsulation, reverse transcription, ddPCR, and bead shrinking, beads were treated with T7 exonuclease to convert the tethered amplicons into single-stranded DNA. To isolate genomic material-containing beads and discard empty ones, the beads were stained with SYBR™ Green I and sorted via FACS. Post-sorting, the SYBR™ Green Lpositive beads were washed with 70% ethanol to remove dye and resuspended in PBS. They were then hybridized with hyb probes tagged with FAM, Cy3, and Cy5 for pol, env, and LTR, respectively. Fig. 5 also shows the sorting of triple-positive beads, with 1.26% of beads exhibiting signals for all three regions, consistent with the expected 1 : 100 ratio of SIV1C to NIH/3T3 cells. Each triple-positive bead was individually sorted into a well and resuspended in a whole transcriptome amplification (WTA) reaction containing forward/reverse primers and KAPA HiFi HotStart ReadyMix. PCR was conducted, and products were purified using 0.8X Ampure XP beads, followed by size validation via Bioanalyzer High Sensitivity DNA Analysis. [071] Fig. 6 shows the application of bead shrinking to magnetically purify cell material inside a hydrogel based on a target nucleic acid. Fig. 6 shows agarose beads attached with a DNA molecule containing a 3 ’-biotin, mixed with agarose beads without attached DNA or biotin. Fig. 6 also shows that the biotin was labelled with streptavidin magnetic nanoparticles. Fig. 6 also shows the purification of agarose-DNA-biotin-Streptavidin-nanoparticle complexes using a magnet. The positive and negative fractions were stained with fluorescent molecules that bind to the nanoparticle complex. Fig. 6 also shows the fluorescence analysis of the negative (flow-through) and positive (column) fractions after magnetic purification by flow cytometer. The negative fraction contained no fluorescent beads, and the positive fraction contains 96% fluorescent beads.

[072] II. Selective magnetic separation of microcompartments containing cells and molecules

[073] The disclosed methods for controlled size and density modulation allow for magnetic separation of hydrogels, wherein shrinking the beads is especially advantageous for high- efficiency magnetic separation.

[074] In an aspect the invention provides methods, materials and systems for the labeling of molecules, cells, or chemicals with magnetic particles within microcompartments and the subsequent isolation of the microcompartment (and its contents) using a magnetic field. The invention provides novel, single-cell genomics and biology techniques for isolating rare cells, molecules, or chemicals.

[075] The invention provides the ability to label and isolate chemical or biochemical reactions that occur within the microcompartments. In one practical application, the invention expands MACS (magnetic activated cell sorting) to any reaction that can be magnetically labeled. For example, we have shown that we can isolate PCR+ compartments.

[076] The invention allows any cell or molecule that can be coupled to the accumulation of magnetic particles within a permeable or semi-permeable compartment (hydrogel, bead, capsule) to be isolated using a magnet. The invention provides an extension of magnetic- activated sorting that enables new applications in single-cell genomics. The critical difference between our invention and currently available methods is the ability to isolate the entire microcompartment based on its contents instead of just the magnetically labeled material.

[077] When combined with our invention, digital PCR can generate amplicons that are labeled with magnetic nanoparticles and allow for the isolation of microcompartments that contain those amplicons. Therefore, our invention provides a way to isolate molecules or cells that contain target RNA or DNA molecules, i.e. a new method of cell cytometry. The invention is highly extensible. Any signal that can be coupled to the accumulation of magnetic particles (including those generated using chemical or biochemical steps) inside a microcompartment (or nanocompartment) can be used. The labeling of compartment material with magnetic particles can occur using a variety of established methods, including protein interactions (e.g. biotinstreptavidin), chemical (e.g. click), nucleic acid hybridization, or non-specific (e.g. hydrophobic interactions).

[078] The invention provides methods, materials and systems for isolating microcompartments (e.g. hydrogel beads or capsules) and their contents using a magnet. After molecules within the microcompartment are labeled, the microcompartment becomes amenable to magnetic -based separation. Microcompartments are flexible picoliter to nanoliter-sized chambers that allow for isolated chemical and biochemical reactions, and may be porous to targeted reaction reagents. Therefore, the ability to generate and isolate magnetic microcompartments extends the power of magnetic-based separations to a broader number of applications in biology and chemistry. One practical application of this technology is the ability to isolate cells that contain specific nucleic acids (DNA or RNA) for sequencing applications. For example, the method can isolate cancer cells based on single base pair changes or isolate cells based on the presence of a virus or other pathogen sequence.

[079] Examples

[080] Enrichment of specific nucleic acids from complex samples. Digital PCR is a widely used tool for molecule counting. A target nucleic acid is amplified within a microcompartment, which is independent of other microcompartments. The number of fluorescent microcompartments, typically microwells or water-in-oil droplets, are counted. Our invention allows clonally amplified molecules within the microcompartments to be easily isolated after digital PCR. This has practical applications for enriching and studying nucleic acids from complex environmental and animal samples. In an embodiment of this application, the target nucleic acid amplification is coupled with magnetic nanoparticle labeling so that only amplified compartments become magnetic with those specific sequences are purified. For example, a forward primer is attached to a hydrogel precursor or a bead, and the amplification reaction uses biotinylated dNTP or biotinylated reverse primer within the microcompartment. After digital PCR, the hydrogel or bead that underwent successful amplification contains biotinylated amplicons. Streptavidin-magnetic particles are added to label the amplified material. These magnetic particles accumulate to high enough concentrations that the microcompartments (and the amplified material) can be purified with a magnet. For examples, this application can enrich and sequence HIV from blood samples, antibiotic resistance (AMR) sequences from environmental samples, or fetal DNA for prenatal testing applications. [081] Magnetic-activated nucleic acid cell sorting. We and others have developed workflows for detecting and sorting single cells based on their nucleic acids using digital droplet PCR. For example, we recently developed a method called FIND-seq (Clark et al, Nature. 2023 Feb;614(7947):326-333.) for sorting cells with HIV provirus. In this workflow, microfluidic devices are used to encapsulate cells within agarose hydrogel beads, trapping and purifying RNA and DNA within the ~50-hydrogel matrix. This material is amenable to ddPCR-based amplification of nucleic acids, labeling with magnetic nanoparticles, and isolation of agarose- containing amplicons. In an embodiment the present invention can isolate cell material based on the amplification of specific nucleic acid sequences, labeling of amplicons with magnetic particles, and the isolation using a magnet of the entire agarose bead containing cell ‘omics (genome, epi-genome, proteome, transcrip tome).

[082] Applications include purifying specific cells containing intracellular pathogens based on pathogen sequences or purifying cancer cells based on genomic mutations. If the starting nucleic acid is at a high enough concentration, no prior amplification is required before labeling and magnetic separation. For example, a multi-copy RNA target (converted to cDNA according to our published protocols) can be labeled with magnetic particles by hybridizing oligo-nucleotides coupled to magnetic particles. Hybridization can employ branched DNA signal amplification techniques (e.g., those used in commercially available RNA-scope). This is an example of magnetic-activated nucleic acid cell sorting.

[083] Purification of beads with specific nucleic acid barcodes. DNA barcodes are common in single -cell genomics workflows. These DNA barcodes are usually attached to hydrogel beads; each bead contains a unique barcoded in high abundance. After single-cell sequencing, reads are grouped by barcode to match sequenced material to individual cells. Often, tens of thousands of single cells and, therefore, tens of thousands of bead barcodes are sequenced. After initial sequencing, specific cells may be identified that are of particular interest. Researchers may wish to re-isolate these specific cells for deeper characterization. To achieve this, magnetic nanoparticles that hybridize to specific barcodes can be used to label those beads for magnetic separation.

[084] In scRNA-seq data from many tissues, abundant cell types utilize the majority of sequencing reads, and most of the money is spent sequencing abundant cells. However, less abundant cells can be equally important in human health. One application of the present invention is to perform shallow sequencing on many cells, identify barcodes from cells of interest from sequencing data, and use magnetic-labeled nucleotides to bind and isolate beads containing those barcodes. This allows cells of interest to be enriched for deep sequencing. Another application applies to multi-omic studies. In this case, two sequencing modalities (e.g. RNA and protein) are linked. This method can select cell barcodes based on one of these sequencing modalities, purify beads with those barcodes, and sequence the second modality. [085] Our method also facilitates the selection of specific chemicals from within DNA- barcoded chemical libraries. In this application, DNA-barcoded chemical libraries are created inside a compartment, the corresponding DNA tag is used to purify chemicals of interest for magnetic separation. This is fundamentally different from a normal magnetic separation because instead of separating an individual chemical by its labeling DNA, this technique labels groups of chemicals contained within or on the scaffold.

[086] CRISPR screens. Forward genetic screens identify the genetic basis of a phenotype. In a typical high throughput CRISPR screen, genetic mutations are made to DNA or RNA using guide RNA and Cas9, and a fluorescent reporter is used to identify the phenotype of interest. Cells containing CRISPR-based changes are sorted based on the reporter, and the guide RNA is sequenced. The abundance of guide RNA sequences is compared between cells with and without the reporter to find guide RNA sequences that modulated that reporter. A common reporter assay utilizes fluorescence so that fluorescence-activated cell sorting (FACS)-based separation can be used. Genetic screens maybe combined with our invention, wherein the reporter can be any amplicon that is generated via digital PCR (e.g. #3). This expands the types of reporters that can be used for CRISPR screens to nucleic acids.

[087] Generation of barcoded beads for single-cell genomics applications. Digital PCR on a single nucleic acid containing conserved regions flanking a barcode (e.g. randomer) can create barcoded beads for single-cell genomics applications. However, single -molecule template loading is random, and occupancy follows the Poisson distribution, requiring the template to be sufficiently dilute to avoid two templates being present in the same microcompartment. This results in many microcompartments with no template, and the resulting bead population mostly (>90%) contains beads with no barcode. Combining our invention with digital PCR can make large numbers of barcoded beads with high barcode diversity and with no non-barcoded beads. Digital droplet PCR on a starting DNA template containing handlei -random barcode-handle2 using handlei and handle2 primers can amplify the barcode so that each bead contains large numbers of the same unique barcode. Templates can have many N’s (e.g., 20N), resulting in massive barcode diversity (4 N = — 1 12 barcodes) following PCR. This removes the need for costly split-pool synthesis of barcodes. As described above, these amplicon-positive beads can be isolated by magnetic labeling and isolation, followed by the release of the magnetic particle by denaturation of the biotinylated strand. These barcoded beads can be used for single-cell genomics applications instead of beads generated by split pool synthesis. [088] References

[089] Clark, I.C., et al., Microfluidics-free single-cell genomics with templated emulsification. Nature Biotechnology, 2023. 41(11): p. 1557-1566.

[090] Clark IC, et al.. Identification of astrocyte regulators by nucleic acid cytometry. Nature.

2023 Feb;614(7947):326-333. doi: 10.1038/s41586-022-05613-0. Epub 2023 Jan 4. PMID: 36599367; PMCID: PMC9980163.

[091] Clark IC, et al. HIV silencing and cell survival signatures in infected T cell reservoirs. Nature. 2023 Feb;614(7947):318-325. doi: 10.1038/s41586-022-05556-6. Epub 2023 Jan 4. PMID: 36599978; PMCID: PMC9908556.

[092] Single Cell Genomic Sequencing Using Hydrogel Based Droplets. F Lan, B Demaree, I Clark, AR Abate. US Patent App. 16/468,652

[093] Hydrogel Purification Of Cell Materials For Per- Activated Cell Sorting. Adam Abate, Iain Clark, Eli Boritz. Filed through UCSF & CZBiohub. CZB-271F-P1. 63/386,745.

Claims

1 . A method for uniformly reducing the size of hydrogel microbeads (HMBs), comprising controlled evaporation of solvent from HMBs through an oil carrier phase, wherein the hydrogel shrinks, and the shrinking hydrogel increases in density until it reaches a density equal to the carrier phase and becomes neutrally buoyant, at which point the HMBs sink and no longer change significantly in size, wherein the evaporation continues until the HMBs reach uniform size, wherein the volume reduction is controllable by modulating the oil and aqueous phases (e.g. by adjusting the initial density of the hydrogel solution or by altering the density of the oil).

2. A method of claim 1 , substantially as shown in Fig. 1.

3. A method of claim 1, substantially as shown in Fig. 1, and comprising controlled evaporation of solvent from HMBs through an oil shell, wherein the hydrogel shrinks, and the shrinking hydrogel increases in density until it reaches a density equal to the oil phase and becomes neutrally buoyant, wherein initially, the beads are placed in a denser-than-water oil phase, such as HFE-7500 with a surfactant or any commercially available oil suitable for droplet generation, the mixture is vortexed with sufficient energy to disperse the beads within the oil, resulting in an emulsion where each droplet encapsulates a single bead, this emulsion is then heated (e.g. to approximately 60 °C for agarose hydrogels), while airflow and oil are continuously supplied during heating to facilitate evaporation and replenish the reduced oil phase, and additionally, the emulsion is intermittently stirred gently, wherein evaporation primarily occurs at the droplets on the top layer — at the interface between oil and air — causing them to diminish in size, wherein this reduction in volume leads to an increase in both the hydrogel concentration and the density of the droplets, wherein intermittent stirring disrupts the settled droplets, ensuring their homogeneous suspension in the oil, wherein the increased density causes the shrunken droplets to reach a density similar to that of the oil, which prevents them from ascending to the air-oil interface to undergo further evaporation, wherein eventually, once all the droplets attain a certain density level, the top emulsion layer dissipates, and the droplets, now with a density matching that of the oil, are uniformly suspended within the oil phase, wherein with the droplets no longer at the air-oil interface, evaporation and subsequent shrinking cease, wherein this process of consistent density equilibrium effectively and uniformly reduces the size of the HMBs by using controlled evaporation and density equalization within the oil phase.

4. A method of claim 1, using an apparatus substantially as shown in Fig. 2.

5. A method of claim 1, wherein the method further comprises FACS-based sorting of the resultant reduced-sized hydrogel microbeads, wherein the bead shrinking may be applied independently of magnetic labeling.

6. A method of claim 1, further comprising enriching the resultant reduced-sized (shrunken) hydrogel microbeads on a FACS machine, substantially as shown in Fig. 4.

7. A method of claim 1, further comprising FACS-based sorting of the resultant reduced-sized hydrogel microbeads, and selective isolation of a transcriptome, such as single-cell RNA sequence a rare cell population, substantially as shown in Fig. 5.

8. A method of claim 1, further comprising deploying the resultant uniformly reduced size hydrogel microbeads (HMBs) for magnetic separation of hydrogels.

9. A method of claim 1 , further comprising deploying the resultant uniformly reduced size hydrogel microbeads (HMBs) in a method for selective magnetic separation of cells or molecules in microcompartments, substantially as shown in Fig. 7, Fig. 8 or Fig. 9.

10. A method for selective magnetic separation of cells or molecules in microcompartments, substantially as shown in Fig. 7, Fig. 8 or Fig. 9.

11. A method of enriching cells, nucleic acids, proteins, or chemicals using magnetic labeling substantially as shown in Fig. 7, and comprising: a) providing microcompartments that are sub-pico to nano-liter vessels created using microfluidic devices or other techniques, wherein the microcompartments can be impermeable or semi-permeable, allowing for the selective retention and labeling of their contents, wherein the microcompartments are loaded with a sample containing target and non-target material, such as wherein the target is a nucleic acid and is amplified to increase the signal; b) linking the target material to a magnetic particle so that the microcompartment with the target material becomes magnetic; c) washing to remove unbound magnetic particles; d) purifying using magnets (e.g. column or tube-based purification) to isolate compartments containing targets of interest, and e) recovering the contents of compartments after purification.

12. A method of magnetic-activated nucleic acid cell sorting to isolate cell genomes and transcriptomes based on nucleic acid biomarkers, substantially as shown in Fig. 8, and comprising: a) providing cells encapsulated within microcompartments and lysed, releasing the genome and transcriptome for biochemical reactions, and adding primers targeting the genome or transcriptome; b) amplifying target nucleic acids using polymerase chain reaction (PCR) or another enzymatic amplification strategy, wherein primers can be modified to enable magnetic labeling, typically using 5 ’ biotin. c) adding affinity streptavidin labeled magnetic nanoparticles to bind to the biotinylated, or functional equivalent, and washing away unbound streptavidin (or functional equivalent) and magnetic nanoparticles; d) placing the solution containing magnetic and non-magnetic microcompartments near a magnet to isolate only magnetic micro compartments, wherein non-magnetic micro compartments are washed away, and the magnet is removed and the microcompartment’s shell or hydrogel is digested or removed to recover cell material of interest.

13. A method of purification of beads with specific nucleic acid barcodes substantially as shown in Fig. 9, and comprising isolating barcoded beads of interest and, therefore, the single-cell material attached to those beads, wherein cDNA reverse transcribed by barcoded primers on the bead is sequenced, and after bioinformatic analysis, cells of interest are identified based on their transcriptional profiles, wherein the barcodes associated with these cells are used to purify the corresponding beads, which can be effected by hybridization of complementary barcodes containing magnetic nanoparticles, magnetic separation, and deep sequencing of the transcriptome, wherein multi-omic embodiments, only one sequencing modality is needed to identify barcodes of interest, for example, in RNA and protein methods (e.g. CITE-seq) or RNA and gRNA methods (Perturb-seq), barcodes can be identified by the transcriptome, followed by barcode-based enrichment using complementary barcodes containing magnetic nanoparticles, wherein the second modality (protein or sgRNA in this example), from only cells of interest, is then sequenced.

14. A method for selective magnetic separation of cells or molecules in microcompartments (flexible picoliter to nanoliter-sized chambers, such as hydrogel beads or capsules), comprising loading a mixture of target and nontarget material into the microcompartments, selectively labeling the target material with a magnetic particle, and magnetically separating the target material from the non-target material to isolate and recover the target material, wherein the method is magnetically separating the microcompartment using the target material, i.e, not just recovering the target, but recovering everything in the microcompartment.

15. A method of any of claims 1-14, further comprising: a) Enrichment of specific nucleic acids from complex samples, e.g. combined with digital PCR to isolate molecules or cells that contain target RNA or DNA sequences; b) Magnetic-activated nucleic acid cell sorting, e.g. isolating cell material based on the amplification of specific nucleic acid sequences, labeling of amplicons with magnetic particles, and the isolation using a magnet of the entire agarose bead containing cell ‘omics (genome, epigenome, proteome, transcriptome); c) Purification of beads with specific nucleic acid barcodes, e.g. enabling selection of specific chemicals from within DNA-barcoded chemical libraries created inside a compartment, wherein the corresponding DNA tag is used to purify chemicals of interest for magnetic separation; d) CRISPR screens, e.g. a reporter assay, wherein the reporter can be any amplicon that is generated via digital PCR; and/or e) Generation of barcoded beads for single-cell genomics applications, e.g. providing large numbers of barcoded beads with high barcode diversity and with no non-barcoded beads, wherein the labeling of compartment material with magnetic particles can occur using a variety of established methods, including protein interactions (e.g. biotin-streptavidin), chemical (e.g. click), nucleic acid hybridization, or non-specific (hydrophobic interactions).