A sticky situation - simple method for rapid poissonian encapsulation of highly aggregation-prone microbeads in polydisperse emulsions

Abstract

Directed evolution leverages the principles of natural selection to engineer biomolecules with desired properties. Microbead-based approaches within water-in-oil emulsions have proven invaluable for high-throughput in vitro selections. However, highly aggregation-prone microbeads present significant challenges, including clustering, inconsistent distribution, and droplet instability. Here, we introduce a simple and cost-effective method for generating polydisperse emulsions with restored Poissonian distributions of highly aggregation-prone microbeads. This approach utilizes modified gel loader pipette tips, drawn out to create nozzles capable of disrupting microbead clusters during emulsification. Two widely utilized oil-surfactant formulations-mineral oil with Abil EM 90 and FluoSurf in HFE 7500 - were evaluated for emulsion preparation. Emulsions prepared using the modified nozzles exhibited exceptional stability, maintaining integrity during week-long incubations at 37°C, and reliably distributed microbeads into droplets in accordance with a Poissonian distribution despite the microbeads' highly aggregation-prone property.

Keywords: aggregation; clustering; emulsion; encapsulation; in vitro compartmentalization; microbeads; poisson distribution.

FIGURE 1

Schematic representation of the b-FAM-A $\beta$ -ATTO643 peptide and the microbead functionalization approach. (a) Chemical structure of the b-FAM-A $\beta$ -ATTO643 peptide. The peptide includes a biotin moiety for microbead attachment and a 5′ FAM fluorescent dye linked via a PEG spacer to the N-terminus, and an ATTO643 dye at the C-terminus. (b) Diagram of the microbead functionalization process, showing streptavidin-coated microbeads conjugated with the b-FAM-A $\beta$ -ATTO643 peptide, which results in exacerbated aggregation behavior.

FIGURE 2

Preparation of modified gel loader pipette tips and their application as nozzles in emulsion generation. (a) Image series of the process used to modify 200 $\mu$ L gel loader pipette tips. The tip is compressed and elongated manually using the back of a scalpel handle, creating a drawn-out nozzle. (b) Image series illustrating the emulsification setup using a modified gel loader pipette tip, where the aqueous phase is gradually added to the oil-surfactant blend during vortexing. (c) Showcase of the modified pipette tip nozzle with its elongated and flattened nozzle design. (d) Stability test of an emulsion prepared using the modified pipette tips. Emulsions appeared intact after 1 week of incubation at 37°C, with no signs of broken droplets and no visible coalescence (see also Supplementary Figure S6) of any released microbeads at the bottom of the tube.

FIGURE 3

Restoration of Poissonian distributions of microbeads in emulsion droplets using gel loader pipette tip-derived nozzles. (a) Microscopic image of a control emulsion (Abil EM 90-based) prepared with unmodified gel loader pipette tips, showing severe clustering of microbeads and a low fraction of droplets containing microbeads. The overview image is captured using DIC microscopy at low magnification (scale bar: 250 $\mu$ m). Zoomed insets display DIC images overlaid with fluorescence (488 and 633 nm excitation, scale bar: 25 $\mu$ m) (b) Microscopic image of an Abil EM 90-based emulsion generated with a modified gel loader pipette tip-derived nozzle, showing a restored Poissonian distribution of microbeads within droplets. Overview DIC image, scale bar 250 $\mu$ m. Zoomed insets, DIC and fluorescence overlays (488 and 633 nm excitation, scale bar: 25 $\mu$ m). (c) Quantitative analysis of microbead encapsulation in Abil EM 90-based emulsion droplets generated using modified gel loader pipette tip-derived nozzles. The fraction of droplets containing n microbeads follows a Poissonian distribution. Error bars represent standard deviation. N = 4 emulsions were prepared with a total of 360 microbead-containing droplets analyzed. Microbead distribution aligned with a Poissonian distribution fit (brown dots). Expected Poisson distributions of microbeads were predicted based on the mean microbead-contianing droplet volume and the total aqueous reagent volume (beige dots). (d) Droplet volume (pL) histogram of microbead-containing droplets in Abil EM 90-based emulsions prepared with modified nozzles. The mean volume is 3.6 pL (orange vertical line). Error bars indicate standard deviation. (e) Microscopic image of a FluoSurf-based emulsion prepared with a gel loader pipette tip-derived nozzle, demonstrating restored Poissonian microbead distributions within droplets. DIC image captured at low magnification (scale bar: 500 $\mu$ m). (f) Quantitative analysis of microbead encapsulation in droplets of FluoSurf-based emulsions generated using the modified gel loader pipette tip nozzles. Shown is the fraction of microbead-bearing droplets containing n amount of microbeads. Error bars denote the standard deviation. N = 3 emulsions were prepared and a total of 1.963 microbead-containing droplets were analyzed. Microbead distribution aligned with a Poissonian distribution fit (brown dots). Expected Poisson distributions of microbeads were predicted based on the mean microbead-contianing droplet volume and the total aqueous reagent volume (beige dots) and based on the droplet volume distribution (orange dots). (g) Histogram of microbead-containing droplet volumes (pL) for FluoSurf-based emulsions prepared with modified pipette tips. The mean volume is indicated by the orange vertical line (8.9 pL). Error bars denote the standard deviation.