Detection and Quantification of Tightly Bound Zn²⁺ in Blood Serum Using a Photocaged Chelator and a DNAzyme Fluorescent Sensor

Abstract

DNAzymes have emerged as a powerful class of sensors for metal ions due to their high selectivity over a wide range of metal ions, allowing for on-site and real-time detection. Despite much progress made in this area, detecting and quantifying tightly bound metal ions, such as those in the blood serum, remain a challenge because the DNAzyme sensors reported so far can detect only mobile metal ions that are accessible to bind the DNAzymes. To overcome this major limitation, we report the use of a photocaged chelator, XDPAdeCage to extract the Zn2+ from the blood serum and then release the chelated Zn2+ into a buffer using 365 nm light for quantification by an 8–17 DNAzyme sensor. Protocols to chelate, uncage, extract, and detect metal ions in the serum have been developed and optimized. Because DNAzyme sensors for other metal ions have already been reported and more DNAzyme sensors can be obtained using in vitro selection, the method reported in this work will significantly expand the applications of the DNAzyme sensors from sensing metal ions that are not only free but also bound to other biomolecules in biological and environmental samples.

Ag/Periodic Mesoporous Organosilica Yolk–Shell Nanostructures for Ratiometric Cu2+ Fluorescent Sensors

“…Fluorescent sensors are frequently employed as practical means of detecting chemicals and ions in the fields of environment and biology. − To meet the growing demand in complex applications, sensors made of multifunctional materials with combined structural and component characteristics, such as thermally activated delayed fluorescence (TADF) sensors and electrogenerated chemiluminescence (ECL) sensors, , have been extensively studied. − Mesoporous silica is regarded as the ideal material to use in the construction of fluorescence sensors because of its benefits of low density, high specific surface area, and flexible modifiability. − Zhang’s group reported a multi-fluorescently traceable system for drug release by using mesoporous silica as a matrix and commercial fluorophores as a signal reference and reporter unit …”

Simultaneous Fe 2+ /Fe 3+ imaging shows Fe 3+ over Fe 2+ enrichment in Alzheimer’s disease mouse brain

“…Among them, RNAcleaving DNAzymes are of particular interest for sensing metal ions because these DNAzymes are often specific for a certain metal ion cofactor (30,32). By conjugating a fluorophore at the end of the enzyme strand, two quenchers at the opposite termini of the enzyme strand and complementary substrate strand, respectively, we take the advantage of melting temperature differences before and after DNAzyme-catalyzed cleavage of the substrate strand and have developed a catalytic beacon approach (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48) that produces metal ion-specific fluorescent turn-on sensors (Fig. 1, A and B).…”

Isothermal Titration Calorimetry for Characterizing the Zinc(II)‐Binding Properties of Photocaged Complexes with Micromolar Affinity

“…We have been building upon our recent reports using decarboxylation as a strategy to design photocages [12][13][14] that has been successfully applied to time-resolved studies in lipid models of cells [15] and quantifying metal ions in blood serum. [16] We are now reporting on the successful design of two decarboxylation photocages that bind Zn 2 + with μM affinity that will expand the scope of possible applications beyond biology and help provide a roadmap for designing effective photocages for metal ions such as Pb 2 + and Hg 2 + that prefer lower coordination numbers and different types of ligands. During our investigations, we developed methods for using isothermal titration calorimetry (ITC) to quantify photocage binding affinities that were unmeasurable by standard techniques.…”