Nanodroplet-Based Reagent Delivery into Water-in-Fluorinated-Oil Droplets

Abstract

In vitro compartmentalization (IVC) is a technique for generating water-in-oil microdroplets to establish the genotype (DNA information)-phenotype (biomolecule function) linkage required by many biological applications. Recently, fluorinated oils have become more widely used for making microdroplets due to their better biocompatibility. However, it is difficult to perform multi-step reactions requiring the addition of reagents in water-in-fluorinated-oil microdroplets. On-chip droplet manipulation is usually used for such purposes, but it may encounter some technical issues such as low throughput or time delay of reagent delivery into different microdroplets. Hence, to overcome the above issues, we demonstrated a nanodroplet-based approach for the delivery of copper ions and middle-sized peptide molecules (human p53 peptide, 2 kDa). We confirmed the ion delivery by microscopic inspection of crystal formation inside the microdroplet, and confirmed the peptide delivery using a fluorescent immunosensor. We believe that this nanodroplet-based delivery method is a promising approach to achieving precise control for a broad range of fluorocarbon IVC-based biological applications, including molecular evolution, cell factory engineering, digital nucleic acid detection, or drug screening.

Keywords: biosensor; microdroplet; microfluidic device; nanodroplet; reagent delivery.

Figure 1

Copper ion delivery into water-in-fluorinated-oil droplets via nanodroplets. (A) Generation of uniform water-in-fluorinated-oil microdroplets by flow-focusing microfluidic device. (B) Microscopy image of microdroplets. Orange arrows indicate the microdroplets containing a microbead. Scale bar, 25 μm. CV: coefficient of variation. (C) Preparation of copper ion nanodroplets by vortexing. (D) Size distribution of the copper ion nanodroplets analyzed by dynamic light scattering. MV: mean volume diameter. SD: standard deviation. (E) Delivery of copper ions into microdroplets through co-incubation leading to crystal formation on microbeads, thereby confirming successful delivery to copper ions.

Figure 2

Confirmation of the copper ion delivery into water-in-fluorinated-oil microdroplets via crystal growth on microbeads. (A) Incubation for 19 h in the absence of copper nanodroplets, 20× objective lens. (B) Incubation for 19 h in the presence of copper nanodroplets, 20× objective lens. (C) Cropped single microdroplet image after 19 h incubation in the presence of copper nanodroplets, 40× objective lens. (D) Incubation for 45 h in the absence of copper nanodroplets, 20× objective lens. (E) Incubation for 45 h in the presence of copper nanodroplets, 20× objective lens. (F) Cropped single microdroplet image after 45 h incubation in the presence of copper nanodroplets, 40× objective lens. Orange arrows indicate the microdroplets containing microbeads under the control condition (without nanodroplets). Blue arrows indicate the droplets showing crystal formed on microbeads. Scale bar for 20× objective lens, 50 μm; scale bar for 40× objective lens, 25 μm.

Figure 3

Nanodroplet-based peptide delivery into water-in-fluorinated-oil microdroplets. (A) Scheme of nanodroplet-based peptide delivery and visualization by immunosensor (Quenchbody). VH and VL, variable region of heavy chain and light chain of antibody. Without peptide, the fluorescence is weak due to the dye–dye quenching and photoinduced electron transfer from tryptophan residues in antibody fragments. The presence of peptide separates fluorophores, leading to a fluorescent signal. (B) Microdroplets containing Quenchbody only (sensor droplets). (C) Microdroplets containing both Quenchbody and 10 μM human p53 peptide (maximum-response droplets; positive control). The maximum-response droplets are spiked into the sensor droplets as internal control during fluorescence imaging. (D) Incubation of mixed microdroplets (90% sensor droplets and 10% maximum-response droplets) in absence of nanodroplets after 3 h. All three microscopic views for this analysis are shown in Figure S1A. (E) Incubation of mixed microdroplets with p53 peptide-containing nanodroplets after 3 h. The orange arrow indicates one of the positive control droplets. The pink arrow indicates one of the sensor droplets after peptide delivery. All three microscopic views for this analysis are shown in Figure S1B. Scale bar, 200 μm. (F) Box plot of fluorescence intensity of the maximum-response droplets after 3 h incubation. (G) Box plot of fluorescence intensity of sensor droplets after 3 h incubation. Box plots indicate the median (center line), mean (cross), first and third quartiles (box edges) and full data ranges (whiskers), and outlier (circles). The level of significance was determined by two-tailed Welch’s t-test.