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Review
. 2024 May 21;96(20):7817-7839.
doi: 10.1021/acs.analchem.4c01510. Epub 2024 Apr 22.

Lab on a Particle Technologies

Rajesh Ghosh  1 , Alyssa Arnheim  1 , Mark van Zee  1 , Lily Shang  1 , Citradewi Soemardy  1 , Rui-Chian Tang  1 , Michael Mellody  1 , Sevana Baghdasarian  1 , Edwin Sanchez Ochoa  1 , Shun Ye  1 , Siyu Chen  1 , Cayden Williamson  1 , Amrith Karunaratne  1 , Dino Di Carlo  1   2   3   4
Affiliations
Review

Lab on a Particle Technologies

Rajesh Ghosh et al. Anal Chem. .
No abstract available

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Conflict of interest statement

The authors declare the following competing financial interest(s): D.D. and the Regents of the University of California have financial interests in Partillion Bioscience which is commercializing Lab on a Particle technology.

Figures

Figure 1
Figure 1
From lab on a chip to lab on a particle. (a) Lab on a chip technology uses microchambers or droplets to confine reactions, enabling the analysis of target cells or molecules with high precision. Parallelization and scale-up rely on the 2D surfaces of chips and custom instrumentation, which often lead to reduced analysis throughput. (b) Lab on a particle technology enables millions of microparticle-based compartments to be scaled in 3D in standard tubes, where fluidic operations are performed using standard laboratory equipment. (c) Operations on particles include cell loading and encapsulation, analyte binding, reagent exchange and washing, and templating of water-in-oil emulsions. Signal enrichment can occur on particles through reactions that are either confined or locally bound. Microparticles are barcoded by shape, size, pattern, color, or other means to enable time-variant analysis as reactions or cell behavior progresses over time. (d) Microparticles are analyzed using standard analytical instruments compatible with cells, such as microscopes, flow cytometers, fluorescence activated cell sorters and single-cell sequencing instruments.
Figure 2
Figure 2
Evolution of cell encapsulation in hydrogel drops for single-cell analysis. (a) Some of the first hydrogel microparticles for cell and protein screening were made using bulk emulsification methods, producing polydisperse agarose particles called gel microdrops (GMDs). [Reprinted with permission from Macmillan Publishers Ltd.: Nature, Weaver, J. C., et al. Nat Biotechnol1988, 6 (9), 1084–1089 (ref (41)) Copyright 1988.] Droplet microfluidics became popular in the early 2000s [Reprinted with permission from ref (42). Anna, S. L., et al. Applied Physics Letters2003, 82 (3), 364–366, 2003 licensed under a Creative Common Attribution (CC BY) license], which was later used to encapsulate cells in uniform GMDs [Reproduced from On-Chip Alginate Microencapsulation of Functional Cells Workman, V, et al. Macromol. Rapid Commun.2008, 29 (2), 165–170 (ref (43)) Copyright 2008 Wiley]. The first studies of aqueous two-phase systems in microfluidic droplets started in the early 2010s. Scale bar is 50 μm. [Reproduced from Vijayakumar, K., et al. Chemical Science2010, 1 (4), 447–452 (ref (44)) with permission from The Royal Society of Chemistry.] This led to the development of hollow hydrogel microparticles that form around encapsulated cells. Scale bar is 50 μm. [Reproduced from Leonaviciene, G., et al. Lab Chip2020, 20 (21), 4052–4062 (ref (45)) with permission from The Royal Society of Chemistry.] (b) Bulk emulsion GMDs: Formation of polydisperse GMDs by vigorously mixing an ungelled polymer solution in oil, to create water-in-oil emulsions. These emulsions, once stabilized, are gelled to form nonuniform GMDs. The oil is removed, and the GMDs are transferred to an aqueous solution. Scale bar is 20 μm. [Reprinted with permission from ref (46). Copyright, 1990 American Society for Microbiology.] (c) Microfluidic GMDs: A microfluidic droplet generator is used to create monodisperse water-in-oil droplet emulsions consisting of ungelled polymer precursors. Subsequently, the solution undergoes gelation to form uniform GMDs. After gelation, the oil is removed, and the GMDs are transferred into an aqueous solution. Scale bar is 100 μm. [Reproduced from Morimoto, Y., et al. Lab Chip2009, 9 (15), 2217–2223 (ref (47)) with permission from The Royal Society of Chemistry.] (d) Core–shell particles: An aqueous two-phase system is employed within a microfluidic droplet generator to produce core–shell microparticles featuring a hollow inner cavity. Polyethylene glycol (PEG), dextran, and a cross-linker are combined to form a water-in-oil emulsion using the microfluidic droplet generator. PEG and dextran undergo phase separation; dextran moves toward the center, while PEG aligns at the surface of the emulsion. Subsequently, the PEG-rich phase is cross-linked to create a solid outer shell. Afterward, the particle is transitioned from oil to water. During this transfer, the inert dextran escapes through the pores of the outer shell, resulting in a hollow interior. Scale bar is 50 μm. [Reproduced with permission from Proceedings of the National Academy of Sciences USA van Zee, M, et al. Proc. Natl. Acad. Sci. U.S.A.2022, 119 (4), e2109430119 (ref (48)).]
Figure 3
Figure 3
Nanovial fabrication and experimental workflow. (a) An aqueous phase consisting of reactive PEG precursor and photoinitiator is coflowed with a second aqueous phase consisting of gelatin or dextran solution in a microfluidics droplet generator resulting in uniform monodispersed aqueous two-phase water-in-oil droplets. The phase-separated droplets are exposed to UV light downstream to polymerize the PEG phase. The dextran or gelatin sacrificial phase is removed during washing steps resulting in an open cavity and final crescent-shaped cross-sectional morphology. (b) If fabricated with gelatin, the nanovials will have a localized gelatin layer at the cavity surface. The gelatin or PEG surface can be functionalized with biotin and streptavidin moieties to attach peptides, proteins, or antibodies to localize cells and their secretions to individual nanovials. (c) Various cell types with a wide diversity of secreted products can be loaded onto and analyzed on nanovials. Cells are loaded onto nanovials in tubes or well plates in bulk, and unbound cells can be filtered out. (d) Fluorescent and/or oligo-barcode labeled detection antibodies are incubated with cells on nanovials to detect their secretions. (e) Single-cell secretion analysis is performed with microscopy, FACS, and/or single-cell sequencing techniques. Scale bar is 100 μm. [First two images reproduced from de Rutte, J., et al. Suspendable Hydrogel Nanovials for Massively Parallel Single-Cell Functional Analysis and Sorting. ACS Nano2022, 16 (5), 7242–7257 (ref (11)) Copyright 2022 American Chemical Society. Last image reprinted with permission from Macmillan Publishers Ltd.: Nature, Udani, S., et al. Nat. Nanotechnol. (ref (9)) Copyright 2023.]
Figure 4
Figure 4
Screening secreted cellular products on nanovials. (a) Antibody secretions can be captured from hybridoma lines, producer cell lines, and primary antibody-secreting B cells. Schematic shows cells are captured, e.g., with antibodies specific to cell surface markers, and secreted antibodies are captured onto antigens or antibodies on the nanovial surface. Images show antigen-specific IgG bound on nanovials (magenta) secreted by HyHEL-5 cells, while 9E10 cells secreting an off-target IgG (blue) do not have corresponding signal on nanovials. Flow scatter plot highlighting the gate used to sort antigen-specific IgG secretors. Presort and postsort microscopy images are shown. The table shows sort enrichment of spiked HyHEL-5 cells. Scale bars are 100 μm. [Reproduced from de Rutte, J., et al. Suspendable Hydrogel Nanovials for Massively Parallel Single-Cell Functional Analysis and Sorting. ACS Nano2022, 16 (5), 7242–7257 (ref (11)). Copyright 2022 American Chemical Society.] (b) Schematic showing multiple cytokines can be captured in parallel from activated T cells that are engaged through TCR interactions with peptides loaded onto class I major histocompatibility complex (p-MHC). Images and FACS plots show T cells captured using p-MHC and screened for IFNγ and TNFα production. Fluorescence peak area vs height scatter plots showing gates used to differentiate nanovial staining vs cell staining of permeabilized cells. Scale bars are 100 μm. [Reprinted with permission from ref (95). Copyright, 2022 D. Koo.] (c) MSCs are captured based on binding to gelatin on nanovials and screened based on extracellular vesicle secretion. Scale bars are 20 μm (top) and 100 μm (bottom). [Reprinted with permission from ref (27). Copyright, 2023 D. Koo.] Imaging flow cytometry of MSCs and captured EVs, stained with an antibody against the tetraspanin, CD9 (red), showing EV secretion positive and negative populations. The viability dye, calcein AM, is used to stain live cells (green). Bottom panel images show calcein AM-stained MSCs (green) on nanovials stained with anti-CD9 (magenta) following FACS sorting based on EV-specific secretion signal gates (low, medium, high secretors). (d) Secretion is associated with single-cell RNA sequencing data (SEC-seq) by using oligo-barcoded detection antibodies and droplet single-cell barcoding of cDNA libraries. Imaging flow cytometry of nonsecreting and IgG-secreting cells. Flow cytometry histograms of VEGF-A signal on nanovials from a VEGF-A concentration sweep or signal from secreting cells over time. Signal is dependent on the presence of a VEGF-A capture antibody. Scale bar is 50 μm. [Reprinted with permission from Macmillan Publishers Ltd.: Nature, Udani, S., et al. Nat. Nanotechnol. (ref (9)). Copyright 2023.] SEC-seq data shows transcriptome-based clustering of single-cell expression profiles and corresponding IgG or VEGF-A secretion signal. [Reprinted with permission from Macmillan Publishers Ltd.: Nature, Cheng, R., et al. Nat. Commun. (ref (26)) Copyright 2023.]
Figure 5
Figure 5
Schematic of spherical particle-templated assays. (a) Particle fabrication methods using microfluidic devices for creating spherical hydrogel microparticles. Commonly used designs include flow focusing and step emulsification devices. Brightfield image of example microparticles produced. (b) Analytes used in particle-templated assays including nucleic acids, proteins, and single cells. (c) Methods for creating particle-templated emulsions using readily available lab instruments, which include vortexing and pipetting, resulting in uniform emulsification. Brightfield image with fluorescent overlay of an example particle-templated emulsion. Scale bars are 100 μm.
Figure 6
Figure 6
Molecular and cellular assays using particle-templated emulsions. (a) Using PTEs to perform droplet digital PCR (ddPCR). The schematic shows droplets positive (green fluorescence) and negative (no fluorescence) for nucleic acid amplification using ddPCR. The fluorescence microscopy images show the amplification of yeast genomic DNA at varying dilutions in a digital regime where the fractions of positive droplets (with fluorescence) correspond with the DNA concentration. Scale bars are 200 μm. [Reproduced from Hatori, M., et al. Particle-Templated Emulsification for Microfluidics-Free Digital Biology. Anal. Chem.2018, 90 (16), 9813–9820 (ref (28)) Copyright 2018 American Chemical Society.] (b) Using PTE to perform ddELISA. The schematic shows droplets positive (green fluorescence) and negative (no fluorescence) for analyte binding and enzymatic amplification using ddELISA. The fluorescence microscopy images illustrate particles (magenta) and fluorescence signal from enzymatic turnover (grayscale) where an increasing fraction of positive droplets corresponds with an increasing concentration of a heart failure protein biomarker. Scale bars are 100 μm. [Reprinted with permission from ref (119). Copyright, 2023 V. Shah.] (c) Using PTE to perform single-cell RNA sequencing. The schematic illustrates coencapsulation of particles and cells in droplets, where color indicates different oligonucleotide barcodes on particles. Similar cell clustering and marker genes are observed for PIP-seq compared to 10X Chromium V3 workflows for cells from healthy breast tissue. [Reprinted with permission from Macmillan Publishers Ltd.: Nature, Clark, I. C., et al. Nat Biotechnol.2023, 41 (11), 1557–1566 (ref (31)). Copyright 2023.]
Figure 7
Figure 7
Workflow for the fabrication and use of shaped particles. (a) Fabrication of shaped microparticles in microfluidic devices using photomasks and UV polymerization. Representative shape-encoded particles used for biomolecular detection. [From Pregibon, D. C., et al. Multifunctional Encoded Particles for High-Throughput Biomolecule Analysis. Science2007, 315 (5817), 1393–1396 (ref (30)) reprinted with permission from AAAS; Kim, L. N., et al. Chem. Commun.2015, 51 (60), 12130–12133 (ref (133)) with permission from The Royal Society of Chemistry; and Destgeer, G., et al. Lab Chip2020, 20 (19), 3503–3514 (ref (22)) with permission from The Royal Society of Chemistry.] (b) Schematic representation of assay workflow using shaped microparticles including incubation and signal amplification steps. (c) Readout of assay results on individual microparticles using standard instrumentation such as microscopes, cell phones, or scanners. [Reproduced from Destgeer, G., et al. Lab Chip2020, 20 (19), 3503–3514 (ref (22)) with permission from The Royal Society of Chemistry; Derveaux, S., et al. Anal. Bioanal. Chem.2008, 391 (7), 2453–2467 (ref (135)) with permission from The Royal Society of Chemistry; and Svedberg, G., et al. Lab Chip2017, 17 (3), 549–556 (ref (134)) with permission from The Royal Society of Chemistry.]
Figure 8
Figure 8
Shaped particle fabrication methods by flow-lithography from oldest to newest. (a) Continuous flow lithography particles. The fabrication system requires a computer-controlled shutter and continuous flowing pumps. Scale bar is 30 μm. [Reprinted with permission from Macmillan Publishers Ltd.: Nature, Dendukuri, D., et al. Nature Mater.2006, 5 (5), 365–369 (ref (10)) Copyright 2006.] (b) Maskless lithography particles. The shutter is replaced with a dynamic mask. Scale bar is 100 μm. [Reprinted with permission from ref (143). Anna, S. L., et al. Applied Physics Letters2003, 82 (3), 364–366 licensed under a Creative Common Attribution (CC BY) license.] (c) Stop flow lithography particles. A computer-controlled valve is introduced to control flow, resulting in higher resolution particles. Scale bar is 30 μm. [Reproduced from Chung, S. E., et al. Applied Physics Letters2007, 91 (4), 041106 (ref (144)) with permission from The Royal Society of Chemistry.] (d) Two-photon continuous lithography particles. This fabrication system requires both a focused laser and a motorized stage. Scale bar is 20 μm. [Reproduced from Stop-Flow Lithography in a Microfluidic Device. Dendukuri, D., et al. Lab Chip20127 (7), 818–828 (ref (145)) Copyright 2012 Wiley.] (e) Inertial flow lithography particles. Before fabrication, the device used to shape flow and the necessary photomask is predesigned using uFlow. Pillars in the device create complex flow geometries, and a shaped photomask produces particles of the desired shape. Scale bar is 100 μm. [Reproduced from Two-Photon Continuous Flow Lithography, Advanced Materials201524 (10), 1304–1308 (ref (146)) Copyright 2015 Wiley.] (f) Coaxial flow lithography particles. A 3D-printed device shapes the coaxial flow. Scale bar is 100 μm. [Reproduced from Destgeer, G., et al. Lab Chip2020, 20 (19), 3503–3514 (ref (22)) with permission from The Royal Society of Chemistry.] Engineering systems and close-up views of particle fabrication region adapted from Lewis, C. L., et al. Anal. Chem.2010, 82 (13), 5851–5858 (ref (142)) with permission from The Royal Society of Chemistry.
Figure 9
Figure 9
Application of shaped microparticles for multiplexed and cellular assays. (a) DNA detection on barcoded particles. The particle is subdivided as follows: graphical barcode portion, detection section for DNA oligomer 1, control (should always be dark), detection for DNA oligomer 2. Particles are approximately 90 μm in width and 180–270 μm in length. [From Pregibon, D. C., et al. Multifunctional Encoded Particles for High-Throughput Biomolecule Analysis. Science2007, 315 (5817), 1393–1396 (ref (30)) reprinted with permission from AAAS.] (b) HPV DNA mutant detection where each differently shaped particle is conjugated to probes for a unique HPV DNA mutant. Images of positive particles corresponding to the barcode for HPV mutant 33. Scale bars are 200 μm. [Reproduced from Kim, L. N., et al. Chem. Commun.2015, 51 (60), 12130–12133 (ref (133)) with permission from The Royal Society of Chemistry.] (c) Images showing miRNA detection (purple) from normal and tumor cells. The oncogenic miRNA, miR-21, is seen in higher concentrations in the particles incubated with tumor lysate. Slightly elevated levels of miR-16, an endogenous standard in miRNA analysis of colon cancer, was seen in the particles incubated with tumor lysate. miR-141 is a marker of poor prognosis associated with advanced colon cancer. The probe cel-miR-54 was used as a negative control. Scale bars are 200 μm. [Reproduced from Derveaux, S., et al. Anal. Bioanal Chem.2008, 391 (7), 2453–2467 (ref (135)) with permission from The Royal Society of Chemistry.] (d) ELISA read out on optical scanner for the detection of autoantibodies from multiple sclerosis patients. ANO2 (1) serves as a positive control for MS autoantibodies, ANO2 (2) serves as negative control, and ZFN688 is a secondary negative control. Particles have a diameter of 900 μm and a thickness of 150 μm. [Reproduced from Svedberg, G., et al. Lab Chip2017, 17 (3), 549–556 (ref (134)) with permission from The Royal Society of Chemistry.] (e) Enzyme-linked assay in particle-templated drops where the fluorescent product within a droplet supported by square particles accumulates while no fluorescent product accumulated in negative control circular particles. Particles range in size from 340–400 μm with cavity dimensions of 100–200 μm. Scale bar is 500 μm. [Reproduced from Destgeer, G., et al. Lab Chip2020, 20 (19), 3503–3514 (ref (22)) with permission from The Royal Society of Chemistry.] (f) MDA-MB-231GFP cells on collagen patterned microcarriers. The first column shows cell proliferation on a microcarrier over time; the second column shows how cells on the nonshelter region were removed via pipetting while cells in the shelter area were protected from pipetting shear forces. The third image shows how the microcarrier orients in flow. Scale bar is 200 μm. [Reprinted with permission from Macmillan Publishers Ltd.: Nature, Wu, C., et al. Microsyst. Nanoeng.2018, 4 (1), 21 (ref (157)) Copyright 2018.] (g) Wrinkled, nonspherical particles. The top row shows two different wrinkled particle shapes, while the bottom row shows cell adhesion differences between a spherical nonwrinkled particle (left) and a wrinkled particle (right). Scale bar is 20 μm (top) and 30 μm (bottom). [Reprinted with permission from Macmillan Publishers Ltd.: Nature, Li, M., et al. Sci. Rep.2016, 6 (1) 30463 (ref (159)) Copyright 2016.] (h) LNCaP cells (magenta) encapsulated in amphiphilic particles. The fluorescence intensity of MMP-cleavable fluorogenic substrate (green) increases with increasing number of encapsulated cells. Scale bar is 200 μm. [From Wu, C., et al. Monodisperse Drops Templated by 3D-Structured Microparticles. Science Advances2020, 6 (45), eabb9023 (ref (12)) reprinted with permission from AAAS.]
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