Citrate-based fluorescent materials for low-cost chloride sensing in the diagnosis of Cystic Fibrosis

Abstract

Chloride is an essential electrolyte that maintains homeostasis within the body, where abnormal chloride levels in biological fluids may indicate various diseases such as Cystic Fibrosis. However, current analytical solutions for chloride detection fail to meet the clinical needs of both high performance and low material or labor costs, hindering translation into clinical settings. Here we present a new class of fluorescence chloride sensors derived from a facile citrate -based synthesis platform that utilize dynamic quenching mechanisms. Based on this low-cost platform, we demonstrate for the first time a selective sensing strategy that uses a single fluorophore to detect multiple halides simultaneously, promising both selectivity and automation to improve performance and reduce labor costs. We also demonstrate the clinical utility of citrate-based sensors as a new sweat chloride test method for the diagnosis of Cystic Fibrosis by performing analytical validation with sweat controls and clinical validation with sweat from individuals with or without Cystic Fibrosis. Lastly, molecular modeling studies reveal the structural mechanism behind chloride sensing, serving to expand this class of fluorescence sensors with improved chloride sensitivities. Thus citrate-based fluorescent materials may enable low-cost, automated multi-analysis systems for simpler, yet accurate, point-of-care diagnostics that can be readily translated into clinical settings. More broadly, a wide range of medical, industrial, and environmental applications can be achieved with such a facile synthesis platform, demonstrated in our citrate-based biodegradable polymers with intrinsic fluorescence sensing.

Figures

Fig. 1. Low-cost synthesis of citrate-based fluorescent sensors. (A) One-pot synthesis scheme of biodegradable photoluminescent polymers (BPLPs), and isolation of CA–cysteine as the chloride recognition element. Chloride sensitivity is observable in a cuvette as drops of NaCl completely quenches the blue fluorescence of CA–cysteine. (B) Facile synthesis scheme of citric acid and various primary amines to expand the family of citrate-based halide sensors with versatile halide sensitivities.

Fig. 2. Fluorescence-based chloride sensing. (A) Chloride quenches CA–cysteine fluorescence strictly under acidic conditions. (B) Stern–Volmer plots linearizing quenching rates over chloride concentration reveal that chloride sensitivity (K SV, or the slope) increases with acidity, R 2 > 0.997. Detection procedures for chloride sensing (shown in schematics) are simplified by standardizing at fixed sulfuric acid concentrations. (C) Comparison of quenching efficiency of common ions, each at 100 mM, in the presence of CA–cysteine at pH 1.3 (except for OH–).

Fig. 3. Clinical validation of sweat chloride for the diagnosis of cystic fibrosis. (A and B) Comparison of CA–cysteine-based and standard clinical mercuric nitrate titration methods to determine sweat chloride for the diagnosis of cystic fibrosis based on 13 volunteers, where correlation is measured by the intraclass correlation coefficient (A) and agreement is measured by a Bland & Altman plot (B).

Fig. 4. Sensing mechanisms. (A) Jablonski diagram summarizing the sensing capabilities and mechanisms of CA–cysteine, in which conditions below pH 2.4 leads to excited state protonation of the 5-carbonyl, forming a cationic state that is key to activating halide quenching processes. (B–C) HOMO (B) and LUMO (C) isosurface plots highlight planarity and an aromatic 2-pyridone element responsible for the bright fluorescence of CA–cysteine.

Fig. 5. Synthesis scheme of citrate-based sensors, and schematics for automated and selective multi-halide sensing. (A–C) Stern–Volmer plots show the chloride sensitivity of CA–cysteine (A), with sensitivity greatly enhanced with CA–cysteamine (B), and greatly decreased with CA–methyl-serine (C) (R 2 > 0.997, R 2 > 0.999, R 2 > 0.995 respectively). (D and E) Bromide and iodide sensitivity of CA–cysteine were catalogued at fixed sulfuric acid concentrations to obtain halide sensitivity under each acidity, which increase with acidity (R 2 > 0.999 for both). (F) Schematic for automated, multi-halide sensing involves measurement of sample at three pH conditions following standardization to solve a system of three Stern–Volmer equations for chloride, bromide, and iodide concentrations.