Luminescent lanthanide probes for cations and anions: Promises, compromises, and caveats.

The long luminescence lifetimes and sharp emission bands of luminescent lanthanide complexes have long been recognized as invaluable strengths for sensing and imaging in complex aqueous biological or environmental media. Herein we discuss the recent developments of these probes for sensing metal ions and, increasingly, anions. Underappreciated in the field, buffers and metal hydrolysis influence the response of many responsive lanthanide probes. The inherent complexities arising from these interactions are further discussed.

Kinetically Inert Macrocyclic Europium(III) Receptors for Phosphate.

The significant role that phosphate plays in environmental water pollution and biomedical conditions such as hyperphosphatemia highlights the need to develop robust receptors that can sequester the anion effectively and selectively from complex aqueous media. Toward that goal, four macrocyclic tris-bidentate 1,2-hydroxypyridonate (HOPO) europium(III) complexes containing either a cyclen, cyclam, TACN, or TACD ligand cap were synthesized and evaluated as phosphate receptors. The solubility of EuIII-TACD-HOPO in water was insufficient for luminescent studies. Whereas EuIII-cyclen-HOPO is eight coordinate with two inner-sphere water molecules, both EuIII-cyclam-HOPO and EuIII-TACN-HOPO are nine coordinate with three inner-sphere water molecules, suggesting that the two coordination states are very close in energy. As observed previously with linear analogues of tripodal HOPO complexes, there is no relationship between the number of inner-sphere water molecules and the affinity of the complex for phosphate. Whereas all three complexes do bind phosphate, EuIII-cyclen-HOPO has the highest affinity for phosphate with the anion displacing both of its inner-sphere water molecules. On the other hand, only one or two of the three inner-sphere water molecules of EuIII-TACN-HOPO and EuIII-cyclam-HOPO are displaced by phosphate, respectively. All three complexes are highly selective for phosphate over other anions, including arsenate. All three complexes are highly stable. EuIII-cyclen-HOPO and, to a lesser extent, EuIII-TACN-HOPO are more kinetically inert than the linear EuIII-Ser-HOPO. EuIII-cyclam-HOPO, on the other hand, is not. This study highlights the significant effect that minor changes in the ligand cap can have on both the ligand exchange rate and affinity for phosphate of tripodal 1,2-dihydroxypyridinonate complexes.

NMR Characterization of Polyethylene Glycol Conjugates for Nanoparticle Functionalization.

The molecular weight, purity, and functionalization of polyethylene glycols are often characterized by 1H NMR spectroscopy. Oft-forgotten, the typical 1H NMR pulse sequence is not 13C decoupled. Hence, for large polymers, the 13C coupled 1H peaks arising from the repeating units have integrations comparable to that of the 1H of the terminal groups. Ignoring this coupling leads to erroneous assignments. Once correctly assigned, these 13C coupled 1H peaks can be used to determine both the molecular weight of the polymer and the efficacy of conjugation of a terminal moiety more accurately than the uncoupled 1H of the repeating unit.

Achieving Selectivity for Phosphate over Pyrophosphate in Ethanol with Iron(III)-Based Fluorescent Probes.

Two iron(III)-based molecular receptors employing 1,2-hydroxypyridinone ligands were developed for phosphate recognition and fluorescence sensing via indicator displacement assay (IDA). The tetra- and pentadentate ligands enable anion recognition by the iron(III) center via its remaining one or two open coordination sites. Weak protective coordination of fluorescein at those sites prevents the formation of µ-oxo dimers in aerated solutions. Its rapid and selective displacement by inorganic phosphate results in a 20-fold increase in the fluorescence of the indicator. Both receptors exhibit high affinity for inorganic phosphate and high selectivity over common competing anions, including halides, acetate, carbonate, and, remarkably, pyrophosphate as well as arsenate. Coordination of phosphate to the iron(III) center was confirmed by ATR-IR and 31P NMR spectroscopy.

Luminescent Lanthanide Probes for Inorganic and Organic Phosphates.

Inorganic and organic phosphates-including orthophosphate, nucleotides, and DNA-are some of the most fundamental anions in cellular biology, regulating numerous processes of both medical and environmental significance. The characteristic long lifetimes of emitting lanthanides, including the brighter europium(III) and terbium(III), make them ideally suited for the development of molecular probes for the detection of phosphates directly in complex aqueous media. Moreover, given their high oxophilicity and the exquisite sensitivity of their quantum yields to their hydration number, those luminescent lanthanides are perfect for the detection of phosphates. Herein we discuss the principles that have guided the recent developments of molecular probes selective for inorganic or organic phosphates and how these lanthanide complexes facilitate the study of numerous biological processes.

Phosphonate coating of commercial iron oxide nanoparticles for nanowarming cryopreserved samples.

New preservation technologies may allow for organ banking similar to blood and biomaterial banking approaches. Using cryoprotective agents (CPAs), aqueous solutions with organic components such as DMSO, propylene glycol, and added salts and sugars, organs can be used to vitrify and store organs at -140 °C. When needed, these organs can be rewarmed in a rapid and uniform manner if CPAs are supplemented with iron oxide nanoparticles (IONPs) in an applied radiofrequency field. Speed and uniformity of warming are both IONP concentration and CPA suspension dependent. Here we present a coating method of small molecule phosphonate linker (PLink) and biocompatible polymer (i.e. polyethylene glycol PEG) that tunes stability and increases the maximum allowable concentration of IONPs in CPA suspension. PLink contains a phosphonate 'anchor' for high irreversible binding to iron oxide and a carboxylic acid 'handle' for ligand attachment. PLink-PEG removes and replaces the initial coating layer of commercially available IONPs (EMG1200 (hydrophobic) and EMG308 (hydrophilic) Ferrotec, Inc., increasing colloidal stability and decreasing aggregation in both water and CPAs, (verified with dynamic light scattering) from minutes (uncoated) to up to 6 days. Heating properties of EMG1200, specific absorption rate (SAR), measured using an applied field of 360 kHz and 20 kA m-1, increased from 20 to 180 W per g Fe with increasing PLink-PEG5000. PEG replacing the initially hydrophobic coating decreased aggregation in water and CPA, consistent with earlier studies on heating performance. Furthermore, although the size is minimized at 0.20 mol PEG per g Fe, heating is not maximized until concentrations above 0.43 mol PEG per g Fe on EMG1200. SAR on hydrophilic EMG308 was preserved at 400 W per g Fe regardless of the amount of PLink added to the core. Herein concentrations of IONP in VS55 (common CPA) significantly above our previous capabilities, sIONP at 10 mg Fe per mL, was reached, 25 mg Fe per mL of 308-PEG5000 and 60 mg Fe per mL of 1200-PEG5000, approaching stock EMG308 in water, 60 mg Fe per mL. Furthermore, at these concentrations cryopreserved Human dermal fibroblast cells were successfully nanowarmed (at applied fields described above), with higher viability as compared to convective rewarming in a water bath and heating rate close to 200 °C min-1, 2.5 times faster than our current system. Using PLink as the coating method allowed for higher concentrations of IONPs to be successfully suspended in CPA without affecting the heating ability. Additionally, the model ligand, PEG, allowed for increased stability over time in nanowarming experiments.

A General Design Strategy Enabling the Synthesis of Hydrolysis-Resistant, Water-Stable Titanium(IV) Complexes.

Despite its prevalence in the environment, the chemistry of the Ti4+ ion has long been relegated to organic solutions or hydrolyzed TiO2 polymorphs. A knowledge gap in stabilizing molecular Ti4+ species in aqueous environments has prevented the use of this ion for various applications such as radioimaging, design of water-compatible metal-organic frameworks (MOFs), and aqueous-phase catalysis applications. Herein, we show a thorough thermodynamic screening of bidentate chelators with Ti4+ in aqueous solution, as well as computational and structural analyses of key compounds. In addition, the hexadentate analogues of catechol (benzene-1,2-diol) and deferiprone (3-hydroxy-1,2-dimethyl-4(1H)-pyridone), TREN-CAM and THPMe respectively, were assessed for chelation of the 45 Ti isotope (t1/2 =3.08 h, ß+ =85 %, Eß+ =439 keV) towards positron emission tomography (PET) imaging applications. Both were found to have excellent capacity for kit-formulation, and [45 Ti]Ti-TREN-CAM was found to have remarkable stability in vivo.

Metallointercalators-DNA Tetrahedron Supramolecular Self-Assemblies with Increased Serum Stability.

Self-assembly of metallointercalators into DNA nanocages is a rapid and facile approach to synthesize discrete bioinorganic host/guest structures with a high load of metal complexes. Turberfield's DNA tetrahedron can accommodate one intercalator for every two base pairs, which corresponds to 48 metallointercalators per DNA tetrahedron. The affinity of the metallointercalator for the DNA tetrahedron is a function of both the structure of the intercalating ligand and the overall charge of the complex, with a trend in affinity [Ru(bpy)2(dppz)]2+ > [Tb-DOTAm-Phen]3+ ≫ Tb-DOTA-Phen. Intercalation of the metal complex stabilizes the DNA tetrahedron, resulting in an increase of its melting temperature and, importantly, a significant increase in its stability in the presence of serum. [Ru(bpy)2(dppz)]2+, which has a greater affinity for DNA than [Tb-DOTAm-Phen]3+, increases the melting point and decreases degradation in serum to a greater extent than the TbIII complex. In the presence of Lipofectamine, the metallointercalator@DNA nanocage assemblies substantially increase the cell uptake of their respective metal complex. Altogether, the facile incorporation of a large number of metal complexes per assembly, the higher stability in serum, and the increased cell penetration of metallointercalator@DNA make these self-assemblies well-suited as metallodrugs.

Exploiting the Fluxionality of Lanthanide Complexes in the Design of Paramagnetic Fluorine Probes.

Fluorine-19 MRI is increasingly being considered as a tool for biomolecular imaging, but the very poor sensitivity of this technique has limited most applications. Previous studies have long established that increasing the sensitivity of 19F molecular probes requires increasing the number of fluorine nuclei per probe as well as decreasing their longitudinal relaxation time. The latter is easily achieved by positioning the fluorine atoms in close proximity to a paramagnetic metal ion such as a lanthanide(III). Increasing the number of fluorine atoms per molecule, however, is only useful inasmuch as all of the fluorine nuclei are chemically equivalent. Previous attempts to achieve this equivalency have focused on designing highly symmetric and rigid fluorinated macrocyclic ligands. A much simpler approach consists of exploiting highly fluxional lanthanide complexes with open coordination sites that have a high affinity for phosphated and phosphonated species. Computational studies indicate that LnIII-TREN-MAM is highly fluxional, rapidly interconverting between at least six distinct isomers. In neutral water at room temperature, LnIII-TREN-MAM binds two or three equivalents of fluorinated phosphonates. The close proximity of the 19F nuclei to the LnIII center in the ternary complex decreases the relaxation times of the fluorine nuclei up to 40-fold. Advantageously, the fluorophosphonate-bound lanthanide complex is also highly fluxional such that all 19F nuclei are chemically equivalent and display a single 19F signal with a small LIS. Dynamic averaging of fluxional fluorinated supramolecular assemblies thus produces effective 19F MR systems.

Design Principles and Applications of Selective Lanthanide-Based Receptors for Inorganic Phosphate.

Phosphate is an anion of both environmental and medical significance. The increase in phosphate levels in surface waters due primarily to run-offs from fertilized agricultural fields causes widespread eutrophication and increasingly large dead-zones. Hyperphosphatemia, a condition in which blood phosphate levels are elevated, is a primary cause of increased mortality and morbidity in chronic or advanced kidney disease. Resolving both of these issues require, in part, new technology that could selectively sequester phosphate in water at neutral pH. The high hydration energy of phosphate, which prevents organic receptors from functioning in water with sufficient affinity, can be overcome via coordination to a hard metal ion. The hardness, oxophilicity and lability of lanthanide ions make them excellent candidates for the design of high affinity phosphate receptors. In this perspective, we discuss how the principles of lanthanide coordination chemistry can be exploited to design sensitive and selective receptors for phosphate. Unlike many supramolecular systems, these hosts do not recognize their anionic guests via directed electrostatic and hydrogen bonding interactions. Instead, the selectivity of our fluxional receptors is governed entirely by acid-base chemistry and electrostatic forces. Parameters that affect the affinity and selectivity of the receptors include the basicities of the coordinating ligand and of the targeted anion, the acidity of the lanthanide ion, and the geometry of the ligand. Uniquely, their affinity for phosphate can be readily tuned by orders of magnitude either by peripheral interactions or by the lanthanide ion itself without affecting their exquisite selectivity over competing anions such as bicarbonate and chloride.

A Walk Across the Lanthanide Series: Trend in Affinity for Phosphate and Stability of Lanthanide Receptors from La(III) to Lu(III).

The trend in affinity of two 1,2-hydroxypyridinonate lanthanide(III) receptors-LnIII-2,2-Li-HOPO and LnIII-3,3-Gly-HOPO (LnIII = LaIII, PrIII, NdIII, SmIII, EuIII, GdIII, TbIII, DyIII, HoIII, ErIII, TmIII, YbIII, and LuIII)-for phosphate across the series was investigated by luminescence spectroscopy via competition against the central europium(III) analog. Regardless of the ligand, the rare earth receptors display a steep and continuous increase in affinity for their phosphate guest across the series, with the later lanthanides displaying the highest affinity for the oxyanion. This trend mirrors that of the stability of the lanthanide receptors, which also increases significantly and continuously from LaIII to LuIII. For these two ligands, the ionic radius of a rare earth, a parameter directly linked to its Lewis acidity, correlates strongly with its affinity for anions, regardless of whether that anion is the one coordinating it (in this case the 1,2-hydroxypyridinonate ligand) or the guest targeted by the lanthanide receptor (in this case phosphate). These observations are indicative of a lack of steric hindrance for coordination of phosphate. Advantageously, increased efficacy of the lanthanide receptor comes with increased stability. The remarkably high stability of LuIII-2,2-Li-HOPO, combined with its high affinity for phosphate, makes it a particularly promising candidate for translational application to medical or environmental sequestration of phosphate since the higher stability will further reduce the risk of the rare earth leaching during anion separation. The unusually large difference in stability between lanthanide complexes (the LuIII complex of 2,2-Li-HOPO is at least 7 orders of magnitude more stable than the LaIII one) bodes well for potential applications in rare earth separation.

Design and Evaluation of the Environmental Outreach Activity for Middle School Students.

The design and development of an outreach activity targeted at 6th grade middle school students, which aims to determine the level of phosphate in samples of water from nearby lakes and streams, are detailed. Several parameters were noted as key to the successful implementation of this activity in a school setting and to it being well-received by both students and teachers. These include the hands-on nature of the experiment, the use of professional scientific equipment and protocols, and the relevance to everyday life and nearby societal issues. Incorporation of the activity into the middle school science curriculum and educational standards are discussed. Qualitative data indicate that the outreach activity was positively received by students and teachers alike. Statistical differences were found between schools and gender even before performing the activity, suggesting that not all student audiences have the same predisposition for science. Overall, the outreach activity appeared to increase stronger endorsements of positive attitudes toward science. Cumulatively, these results suggest that the outreach activity was well-received and engaging and indicate that it increases positive attitudes toward science, though more in-depth and longitudinal analyses are necessary for future studies.

Catechol-Based Functionalizable Ligands for Gallium-68 Positron Emission Tomography Imaging.

Four tris-bidentate catecholamide (CAM) ligands were synthesized, characterized, and evaluated as ligands for radiolabeling of gallium-68 for positron emission tomography (PET). Three of those ligands, 2,2-Glu-CAM, 3,3-Glu-CAM, and TREN-bisGlyGlu-CAM, incorporate ligand caps that contain a pendant carboxylic group for further conjugation to targeting moieties. The acyclic ligands all exhibited high (>80%) radiolabeling yields after short reaction times (<10 min) at room temperature, a distinct advantage over macrocyclic analogues that display slower kinetics. The stabilities of the four GaIII complexes are comparable to or higher than those of other acyclic ligands used for gallium-68 PET imaging, such as desferrioxamine, with pGa values ranging from 21 to >24, although the functionalizable ligands are less stable than the parent GaIII-TREN-CAM. In vivo imaging studies and ex vivo pharmacokinetic and biodistribution studies indicate that the parent [68Ga]Ga-TREN-CAM is stable in vivo but is rapidly cleared in <15 min, probably via a renal pathway. The rapid and mild radiolabeling conditions, high radiolabeling yields, and high stability in human serum (>95%) render TREN-bisGlyGlu-CAM a promising candidate for gallium-68 chelation.

Design and applications of metal-based molecular receptors and probes for inorganic phosphate.

Inorganic phosphate has numerous biomedical functions. Regulated primarily by the kidneys, phosphate reaches abnormally high blood levels in patients with advanced renal diseases. Since phosphate cannot be efficiently removed by dialysis, the resulting hyperphosphatemia leads to increased mortality. Phosphate is also an important component of the environmental chemistry of surface water. Although required to secure our food supply, inorganic phosphate is also linked to eutrophication and the spread of algal blooms with an increasing economic and environmental burden. Key to resolving both of these issues is the development of accurate probes and molecular receptors for inorganic phosphate. Yet, quantifying phosphate in complex aqueous media remains challenging, as is the development of supramolecular receptors that have adequate sensitivity and selectivity for use in either blood or surface waters. Metal-based receptors are particularly well-suited for these applications as they can overcome the high hydration enthalpy of phosphate that limits the effectiveness of many organic receptors in water. Three different strategies are most commonly employed with inorganic receptors for anions: metal extrusion assays, responsive molecular receptors, and indicator displacement assays. In this review, the requirements for molecular receptors and probes for environmental applications are outlined. The different strategies deployed to recognize and sense phosphate with metal ions will be detailed, and their advantages and shortfalls will be delineated with key examples from the literature.

The Ligand Cap Affects the Coordination Number but Not Necessarily the Affinity for Anions of Tris-Bidentate Europium Complexes.

To evaluate the effect of ligand geometry on the coordination number, number of inner-sphere water molecules, and affinity for anions of the corresponding lanthanide complex, six tris-bidentate 1,2-hydroxypyridonate (HOPO) europium(III) complexes with different cap sizes were synthesized and characterized. Wider or more flexible ligand caps, such as in EuIII-TREN-Gly-HOPO and EuIII-3,3-Gly-HOPO, enable the formation of nine-coordinate europium(III) complexes bearing three inner-sphere water molecules. In contrast, smaller or more rigid caps, such as in EuIII-TREN-HOPO, EuIII-2,2-Li-HOPO, EuIII-3,3-Li-HOPO, and EuIII-2,2-Gly-HOPO, favor eight-coordinate europium(III) complexes that have only two inner-sphere water molecules. Notably, there is no correlation between the number of inner-sphere water molecules and the affinity of the Eu(III) complexes for phosphate. Some q = 2 (EuIII-TREN-HOPO, EuIII-3,3-Li-HOPO, and EuIII-2,2-Gly-HOPO) and some q = 3 (EuIII-TREN-Gly-HOPO) complexes have no affinity for anions, whereas one q = 2 complex (EuIII-2,2-Li-HOPO) and one q = 3 complex (EuIII-3,3-Gly-HOPO) have a high affinity for phosphate. For the latter two systems, each inner-sphere water molecule is replaced with a phosphate anion, resulting in the formation of EuLPi2 and EuLPi3 adducts, respectively.

A Combination of Factors: Tuning the Affinity of Europium Receptors for Phosphate in Water.

Although recognition of hard anions by hard metal ions is primarily achieved via direct coordination, electrostatic and hydrogen-bonding interactions also play essential roles in tuning the affinity of such supramolecular receptors for their target. In the case of EuIII hydroxypyridinone-based complexes, the addition of a single charged group (-NH3+, -CO2-, or -SO3-) or neutral hydrogen-bonding moiety (-OH) peripheral to the open coordination site substantially affects the affinity of the metal receptor for phosphate in water at neutral pH. A single primary ammonium increases the first association constant for phosphate in neutral water by 2 orders of magnitude over its neutral analogue. The addition of a peripheral alcohol group also increases the affinity of the receptor but to a lesser degree (21-fold). On the other hand, negatively charged complexes bearing either a carboxylate or sulfate moiety have negligible affinity for phosphate. Interestingly, the peripheral group also influences the stoichiometry of the lanthanide receptor for phosphate. While the complex bearing a -NH3+ group binds phosphate in a 1:2 ratio, those with -OH and H (control) both form 1:3 complexes. Although the positively charged EuIII-Lys-HOPO has the highest Ka1 for phosphate, a greater increase in luminescence intensity (36-fold) is observed with the neutral EuIII-Ser-HOPO complex. Notably, whereas the affinity of the EuIII complexes for phosphate is substantially influenced by the presence of a single charged group or hydrogen-bond donor, their selectivity for phosphate over competing anions remains unaffected by the addition of the peripheral groups.

The Stability of the Complex and the Basicity of the Anion Impact the Selectivity and Affinity of Tripodal Gadolinium Complexes for Anions.

The affinities and selectivities of lanthanide complexes with open coordination sites for anions vary considerably with the chelate. In order to determine the effect of the stability of a lanthanide complex on its affinity for anions, five different complexes featuring different bidentate chelating moieties were synthesized, and their affinity for anions in water at neutral pH were evaluated by longitudinal relaxometry measurements. The chelates comprise both oxygen and nitrogen donors including maltol, 1,2-hydroxypyridinone, hydroxamic acid, pyridin-2-ylmethanol, and carbamoylmethylphosphonate diester. They were chosen to span a range of basicities all the while maintaining a similar tripodal tris-bidentate architecture, thereby allowing for a direct study of the role of the coordinating motif on the supramolecular recognition of anions by the corresponding GdIII complex. Overall, for ligands containing the same number of protonation steps, and therefore the same charge at neutral pH, the lower the acidity of the chelate (higher ∑pKa's), the less stable the corresponding GdIII complex, and the higher its affinity for anions. Regardless of the number of protonation steps, the more stable GdIII complexes form ternary or quaternary assemblies with coordinating anions. In contrast, the same anions readily displace the chelate of the least stable complexes, resulting instead in the formation of GdIII·anion precipitates. Irrespective of the chelate, in the absence of steric hindrance at the open coordination site, the affinity of GdIII complexes for anions follows the order phosphate > arsenate > bicarbonate > fluoride. Hence, the selectivity and affinity of GdIII complexes of tripodal tris-bidentate chelates for anions is a function of the stability of the GdIII complex and the basicity of the anion.

A turn-on luminescent europium probe for cyanide detection in water.

A luminescent europium probe that responds to cyanide directly in water with a large nine-fold turn-on of the EuIII centered time-gated luminescence is presented. Unlike other CN- probes reported, the mechanism of action of EuIII-Lys-HOPO does not rely on reaction of CN- with the probe, but on direct coordination of CN- to the EuIII ion concomitant with displacement of three inner-sphere water molecules. This unusual coordination of CN- with a lanthanide ion in aqueous solution was confirmed by luminescence lifetime measurements.

Fluorinated Paramagnetic Complexes: Sensitive and Responsive Probes for Magnetic Resonance Spectroscopy and Imaging.

Fluorine magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) of chemical and physiological processes is becoming more widespread. The strength of this technique comes from the negligible background signal in in vivo19F MRI and the large chemical shift window of 19F that enables it to image concomitantly more than one marker. These same advantages have also been successfully exploited in the design of responsive 19F probes. Part of the recent growth of this technique can be attributed to novel designs of 19F probes with improved imaging parameters due to the incorporation of paramagnetic metal ions. In this review, we provide a description of the theories and strategies that have been employed successfully to improve the sensitivity of 19F probes with paramagnetic metal ions. The Bloch-Wangsness-Redfield theory accurately predicts how molecular parameters such as internuclear distance, geometry, rotational correlation times, as well as the nature, oxidation state, and spin state of the metal ion affect the sensitivity of the fluorine-based probes. The principles governing the design of responsive 19F probes are subsequently described in a "how to" guide format. Examples of such probes and their advantages and disadvantages are highlighted through a synopsis of the literature.

Development of a Click-Chemistry Reagent Compatible with Mass Cytometry.

The recent development of mass cytometry has allowed simultaneous detection of 40 or more unique parameters from individual single cells. While similar to flow cytometry, which is based on detection of fluorophores, one key distinguishing feature of mass cytometry is the detection of atomic masses of lanthanides by mass spectrometry in a mass cytometer. Its superior mass resolution results in lack of signal overlap, thereby allowing multiparametric detection of molecular features in each single cell greater than that of flow cytometry, which is limited to 20 parameters. Unfortunately, most detection in mass cytometry relies on lanthanide-tagged antibodies, which is ideal to detect proteins, but not other types of molecular features. To further expand the repertoire of molecular features that are detectable by mass cytometry, we developed a lanthanide-chelated, azide-containing probe that allows click-chemistry mediated labeling of target molecules. Following incorporation of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) during DNA synthesis in S-phase of the cell cycle, we demonstrate that the probe introduced here, tagged with Terbium-159 (159Tb), reacts via copper-catalyzed azide-alkyne Huisgen cycloaddition (click-chemistry) with Edu. Thus, detection of 159Tb makes it possible to measure DNA synthesis in single cells using mass cytometry. The approach introduced here shows similar sensitivity (true positive rate) to other methods used to measure DNA synthesis in single cells by mass cytometry and is compatible with the parallel antibody-based detection of other parameters in single cells. Due to its universal nature, the use of click-chemistry in mass cytometry expands the types of molecular targets that can be monitored by mass cytometry.