Ultra‐fast Cycling for Multiplexed Cellular Fluorescence Imaging

Abstract

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Herein, we describe an ultra‐fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz)/trans‐cyclooctene (TCO)‐mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 <1 s) than predicted. This allowed multi‐cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.

Cellular point-of-care diagnostics using an inexpensive layer-stack microfluidic device

“…19 Cyclic imaging, i.e., repeat staining and destaining of scant cellular specimen, allows much deeper biomarker analysis based on diverse immune and cancer markers. 3,20,21 Manual handling of such samples, however, is cumbersome and labor-intensive, a reason for which we explored microfluidic systems. An overarching goal of this work is to develop inexpensive designs that could be readily employed in LMIC and other resource-limited settings.…”

“…FAST probes are designed to include a modular linker between fluorochromes and antibodies with an TCO molecule for clicking with a tetrazine-quencher. FAST probes were synthesized as described in detail in our previous study, 3 stored as the carboxylic acids, and activated for antibody labeling with our in situ NHS/TFP activation chemistry. The dTCO-PEG 6 -CO 2 H blocking reagent was synthesized from dTCO-PNP and amino-dPEG 6 -CO 2 H and characterized by LC-MS. All reagents were obtained from commercial sources: fluorophores were purchased from Click Chemistry Tools or Fluoroprobes; BHQ®-3 Amine from LGC Biosearch Technologies; N -α-Boc- N -ε-Fmoc-lysine from Chem-Impex; Amino-dPEG® n -carboxylic acids ( n = 4,6) from Quanta BioDesign; dry solvents and coupling reagents from Sigma Aldrich.…”

“…Antibodies were purchased as carrier-free for modification with FAST probes as previously described. 3 Antibodies were exchanged into bicarbonate buffer (pH 8.4) using a 40 k Zeba column (Thermo Fisher) and incubated with a 5- to 10-fold molar excess of the FAST probe with 10% DMSO for 25 minutes at room temperature in dark. After conjugation reaction, unbound FAST probes were removed by another 40 k Zeba column equilibrated with PBS.…”

“…We utilized FAST imaging, which exploits highly accelerated bioorthogonal tetrazine/trans-cyclooctene (Tz/TCO) click reaction to enable iteration of rapid quenching and re-staining of immunofluorescence as described in our previous studies. 3,4 FAST probes were synthesized with a ternary scaffold containing a fluorophore (AF488, AF555, or AF647), a TCO for clicking with a Tz-labeled quencher (black hole quencher, BHQ3), and a NHS/TFP-ester for reaction with aminecontaining molecules such as antibody. Cells were stained with antibodies conjugated to the FAST probes and the FAST fluorophores were quenched using Tz-BHQ3 in between cycles.…”

“…1,2 Recent research has focused on developing automated pipelines for comprehensive analysis of FNA tumor samples. This has included development of cyclic staining methods for high multiplexing, 3,4 automated imaging systems 5 and AI-enabled software. 6 One missing piece to the puzzle, however, has been the development of robust sample handling methods for cell immobilization and repeat staining cycles.…”