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

Abstract

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...

Nanodroplet-based reagent delivery into water-in-fluorinated-oil droplets

“…The dispersed phase containing the human p53 protein fluorescent immunosensor (p53 Quenchbody) was used to make microdroplets for reagent delivery assessment of middle-size biomolecules. 17 The sensor microdroplets were defined as the ones containing the p53 Quenchbody but without p53 peptide, which had low background fluorescence intensity (Fig. 3B).…”

“…The p53 Quenchbody immunosensor was prepared as described in a previous study. 17 The Quenchbody immunosensor microdroplets were generated on a flow-focusing microfluidic chip with a nozzle size of 20 μm. The disperse phase of 60 nM p53 Quenchbody in PBS buffer (pH 7.4) was loaded into PTFE tubing (inner diameter 0.8 mm) and driven by a syringe pump NE-1000 (New Era, Farmingdale, NY, USA).…”

“…Similarly, we also confirmed the delivery of the 20 amino acid human p53 peptide by visualizing the fluorescent signals in water-in-fluorinated-oil microdroplets containing the p53 fluorescent immunosensor. 17 We revealed that the nanodroplet-based reagents delivery was a promising approach for metal ions and middle-size biomolecules delivery into water-in-fluorinated-oil microdroplets. It has a great potential to be used for performing multi-step cellular and biochemical assays within water-in-fluorinated-oil microdroplets such as synchronously triggering the alkyne-azide click reaction 18,19 , activation of enzymes 20,21 , or the inhibition of enzymatic reactions 22,23 in water-influoronated-oil droplets.…”

The Dawn of a New Era: Targeting the “Undruggables” with Antibody-Based Therapeutics

“…Unlike conventional antibody-based probes, quenchbodies have the advantage of antigen-dependent signal generation. To date, more than ten quenchbodies targeting various proteins and small molecules have been developed. ,− Very recently, an anti - p53 quenchbody was constructed and successfully used for live-cell imaging of intracellular POIs after being delivered into live cells by electroporation . Of note, the fluorescence change of this strategy was highly dependent on the amino acid sequence and three-dimensional structure of the mAb.…”

“…441,451−455 Very recently, an anti-p53 quenchbody was constructed and successfully used for live-cell imaging of intracellular POIs after being delivered into live cells by electroporation. 456 Of note, the fluorescence change of this strategy was highly dependent on the amino acid sequence and three-dimensional structure of the mAb. As difference exists in the number and distribution of Trp residues in different antibodies, genetic engineering was often needed to obtain optimal quenching efficiency.…”