Development of Blood‐Cell‐Selective Fluorescent Biodots for Lysis‐Free Leukocyte Imaging and Differential Counting in Whole Blood

Abstract Complete blood count with leukocyte (white blood cell, WBC) differential is one of the most frequently ordered clinical test for disease diagnosis. Herein, multifunctional fluorescent carbon dots derived from biomolecules (biodots) for rapid lysis‐free whole blood analysis are developed. Specifically, two types of biodots are molecularly engineered through hydrothermal synthesis for differential blood cells labeling. Type I biodots synthesized from amino acid (serine/threonine) precursors and passivated with polyethylenimine can label both red blood cells (RBCs) and WBCs with excellent contrast in fluorescence intensity, enabling direct counting of leukocytes in whole blood samples without a tedious RBC lysis step. It also allows three‐part leukocyte differential counting by flow cytometry without using expensive fluorophore‐conjugated antibodies. On the other hand, Type II biodots synthesized from the same amino acid precursors but passivated with a biopolymer (chitosan) are able to selectively lyse RBCs with greater than 98% efficiency to allow simultaneous fluorescent labeling of leukocytes for WBC counting in whole blood. It is envisioned that these novel nanoreagents, which eliminate the cumbersome lysis and antibody conjugation steps for selective labeling of different blood cells, would revolutionize disease diagnostics toward achieving faster, cheaper, and more accurate whole blood analyses in one test.