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

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.

Visualizing intracellular target antigens in live cells.

In order to further visualize intracellular dynamics, precise imaging of endogenous proteins in live cells was performed using an antigen-binding fragment (Fab)-based Quenchbody (Q-body). The transfected Q-body probe showed an antigen-dependent fluorescence response, enabling the clear visualization and sorting of cells expressing p53, a tumor suppressor biomarker.

Rapid and sensitive SARS-CoV-2 detection using a homogeneous fluorescent immunosensor Quenchbody with crowding agents.

Antigen tests for SARS-CoV-2 are widely used by the public during the ongoing COVID-19 pandemic, which demonstrates the societal impact of homogeneous immunosensor-related technologies. In this study, we used the PM Q-probe and Quenchbody technologies to develop a SARS-CoV-2 nucleocapsid protein (N protein) homogeneous immunosensor based on a human anti-N protein antibody. For the first time, we uncovered the crowding agent's role in improving the performance of the double-labeled Quenchbody, and the possible mechanisms behind this improvement are discussed. The 5% polyethylene glycol 6000 significantly improved both the response speed and sensitivity of SARS-CoV-2 Quenchbodies. The calculated limit of detection for recombinant N protein was 191 pM (9 ng mL-1) within 15 min of incubation, which was 9- to 10-fold lower than the assay without adding crowding agent. We also validated the developed immunosensor in a point-of-care test by measuring specimens from COVID-19-positive patients using a compact tube fluorometer. In brief, this work shows the feasibility of Quenchbody homogeneous immunosensors as rapid and cost-efficient tools for the diagnosis and high-throughput analysis of swab samples in large-scale monitoring and epidemiological studies of COVID-19 or other emerging infectious diseases.

Intra Q-body: an antibody-based fluorogenic probe for intracellular proteins that allows live cell imaging and sorting.

Although intracellular biomarkers can be imaged with fluorescent dye(s)-labeled antibodies, the use of such probes for precise imaging of intracellular biomarkers in living cells remains challenging due to background noise from unbound probes. Herein, we describe the development of a conditionally active Fab-type Quenchbody (Q-body) probe derived from a monoclonal antibody (DO-1) with the ability to both target and spatiotemporally visualize intracellular p53 in living cells with low background signal. p53 is a key tumor suppressor and validated biomarker for cancer diagnostics and therapeutics. The Q-body displayed up to 27-fold p53 level-dependent fluorescence enhancement in vitro with a limit of detection of 0.72 nM. In fixed and live cells, 8.3- and 8.4-fold enhancement was respectively observed. Furthermore, we demonstrate live-cell sorting based on p53 expression. This study provides the first evidence of the feasibility and applicability of Q-body probes for the live-cell imaging of intrinsically intracellular proteins and opens a novel avenue for research and diagnostic applications on intracellular target-based live-cell sorting.

Tissue Extract Fractions from Starfish Undergoing Regeneration Promote Wound Healing and Lower Jaw Blastema Regeneration of Zebrafish.

Natural bioactive materials provide an excellent pool of molecules for regenerative therapy. In the present study, we amputate portions of the arms of Archaster typicus starfish, extract and separate the active biomaterials, and compare the effects of each fraction on in vitro wound healing and in vivo lower jaw regeneration of zebrafish. Compared with crude extract, normal hexane fractions (NHFs) have a remarkable effect on cellular proliferation and collective migration, and exhibit fibroblast-like morphology, while methanol-water fractions (MWFs) increase cell size, cell-cell adhesion, and cell death. Relative to moderate mitochondrialand lysosomal aggregation in NHFs-cultured cells, MWFs-cultured cells contain more and bigger lysosomal accumulations and clump detachment. The in vivo zebrafish lower jaw regeneration model reveals that NHFs enhance blastema formation and vasculogenesis, while MWFs inhibit fibrogenesis and induce cellular transformation. Gene expression analyses indicate that NHFs and MWFs separately activate blastema-characteristic genes as well as those genes-related to autophagy, proteasome, and apoptosis either during cell scratch healing or ganciclovir-induced apoptosis. Our results suggest that bioactive compounds from NHFs and MWFs could induce blastema formation and remodeling, respectively, and prevent tissue overgrowth.