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Counting and recognizing single bacterial cells are crucial to the diagnosis of bacterium-induced disease and study of cell-to-cell variability as well as the related antibiotic resistance mechanism. A higher sensitive and selective method has always been desired for a more accurate single bacterial cell analysis. We report a lanthanide-encoding inductively coupled plasma (ICP) mass spectrometric approach for counting and recognizing single bacterial cells for the first time. When noncanonical alkyne-d-alanine (aDA) was added to five typical bacterial strains of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Shigella dysenteriae, and Vibrio parahemolyticus, aDA was metabolically assembled into the peptidoglycan layer-supported bacterial cell wall followed by post-clickable europium-tagging with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-azidopropyl ethylacetamide-europium complex (azide-DOTA-Eu). Such Eu-tagged bacterial cells can be deemed as Eu-engineered particles, delivering more than 5 orders of magnitude self-signal-amplification outcome relative to the single bacterial cells themselves when 151/153Eu is determined by single particle ICP mass spectrometry (spICPMS). This metabolic assembly of aDA mediated Eu-encoding signal amplification strategy breaks through the detection limit of spICPMS and ensures that we directly count a single bacterial cell. The individual bacterial strains we counted can be simultaneously recognized through their corresponding lanthanide (Ln)-coded polyclonal antibody (Ln = 139La, 141Pr, 142Nd, 152Sm, and 160Gd, respectively), serving as a specific bacterial identification (Ln-pAb-ID). Moreover, the developed approach was applied to show the different behavior between genetically identical Staphylococcus aureus under the treatments of vancomycin and Ag nanoparticles, demonstrating that such a lanthanide-encoding spICPMS approach provided a new way to discover still ambiguous cell-to-cell variability.

Although we believe that the cell surface sialic acids (Sias) are playing an important role in cell-cell interactions and related tumor metastasis processes, acquisition of their quantitative information has yet been a challenge to date. Here, we reported the construction of a new analytical platform for Sias-specific imaging and quantification. We used N-azidoacetyl-mannosamine tetraacylated as a metabolic sugar substrate to bioassemble azido-Sias on the surface of cells via the metabolic pathway of Sias de novo synthesis. These azido-Sias allow us to perform a duplex Sias-specific analysis with various fluorescent and elemental reporters such as DIBO-Alexa Fluor 647, DBCO-DOTA-Eu, and DBCO-PEG-BODIPY, which can be easily labeled and/or tagged through an effective copper-free bioorthogonal click reaction. Compared to the previous reported strategies, we quantified the cell surface Sias with the LODs (3σ) down to 8.9 fmol and 0.24 pmol using Eu- andB-species unspecific isotope dilution ICPMS, in addition to their red- and green-CLSM profiling. Such a platform enables us to evaluate Sias regulation under the administration of paclitaxel, finding that 1 μM paclitaxel induced a significant Sias decrease of 67% on the surface of hepatic tumor cell SMMC-7721, while had no obvious adverse effect to that of para-carcinomatous liver cell LO2. Besides Sias, we believe that this metabolism-based click-mediated platform will provide opportunities to study other monosaccharides and their corresponding biological roles when more corresponding chemically modified sugar substrates and specific bioorthogonal reactions are developed.

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