DNAzymes have been shown as a promising platform for metal ions detection and a few DNAzyme-based sensors have been reported to detect metal ions inside cells. However, these methods required an influx of metal ions to increase their concentrations for detection. To address this major issue, we herein report the design of a catalytic hairpin assembly (CHA) reaction to amplify the signal from photocaged Na+-specific DNAzyme to detect endogenous Na+ inside cells. Upon light activation and in the presence of Na+, NaA43 DNAzymes cleave the substrate strands and release initiator DNA that trigger the followed CHA amplification reaction. This strategy has allowed detection of endogenous Na+ inside cells, which has been demonstrated by both fluorescent imaging of individual cells and flow cytometry of the whole cell population. This method can be generally applied to detect other endogenous metal ions and thus contribute to deeper understanding of the role of metal ions in biological systems.

Viral infections are a major global health issue, but no current method allows rapid, direct, and ultrasensitive quantification of intact viruses with the ability to inform infectivity, causing misdiagnoses and spread of the viruses. Here, we report a method for direct detection and differentiation of infectious from noninfectious human adenovirus and SARS-CoV-2, as well as from other virus types, without any sample pretreatment. DNA aptamers are selected from a DNA library to bind intact infectious, but not noninfectious, virus and then incorporated into a solid-state nanopore, which allows strong confinement of the virus to enhance sensitivity down to 1 pfu/ml for human adenovirus and 1 × 10 4 copies/ml for SARS-CoV-2. Applications of the aptamer-nanopore sensors in different types of water samples, saliva, and serum are demonstrated for both enveloped and nonenveloped viruses, making the sensor generally applicable for detecting these and other emerging viruses of environmental and public health concern.

CONSPECTUS: Metal ions can be beneficial or toxic depending on their identity, oxidation state, and concentration. Therefore, the ability to detect and quantify different types of metal ions using portable sensors or in situ imaging agents is important for better environmental monitoring, in vitro medical diagnostics, and imaging of biological systems. While numerous metal ions in different oxidation states are present in the environment and biological systems, only a limited number of them can be detected effectively using current methods. In this Account, we summarize research results from our group that overcome this limitation by the development of a novel class of activity-based sensors based on metal-dependent DNAzymes, which are DNA molecules with enzymatic activity. First, we have developed an in vitro selection method to obtain DNAzymes from a large DNA library of up to 10 15 sequences that can carry out cleavage of an oligonucleotide substrate only in the presence of a specific metal ion with high selectivity. Negative selection steps can further be used to improve the selectivity against potentially competing targets by removing sequences that recognize the competing metal ions. Second, we have developed a patented catalytic beacon method to transform the metal-dependent DNAzyme cleavage reaction into a turn-on fluorescent signal by attaching a fluorophore and quenchers to the DNAzyme complex. Because of the difference in the melting temperatures of DNA hybridization before and after metal-ion-dependent cleavage of the DNAzyme substrate, the fluorophore on the DNA cleavage product can be released from its quenchers to create a turn-on fluorescent signal. Because DNAzymes are easy to conjugate with other signaling moieties, such as gold nanoparticles, lanthanide-doped upconversion nanoparticles, electrochemical agents, and gadolinium complexes, these DNAzymes can also readily be converted into colorimetric sensors, upconversion luminescence sensors, electrochemical sensors, or magnetic resonance contrast agents. In addition to describing recent progress in developing and applying these metal ion sensors for environmental monitoring, point-of-care diagnostics, cellular imaging, and in vivo imaging in zebrafish, we summarize major advantages of this class of activity-based sensors. In addition to advantages common to most activity-based sensors, such as enzymatic turnovers that allow for signal amplification and the use of initial rates instead of absolute signals for quantification to avoid interferences from sample matrices, the DNAzyme-based sensors allow for in vitro selection to expand the method to almost any metal ion under a variety of conditions, negative selection to improve the selectivity against competing targets, and reselection of DNAzymes and combination of active and inactive variants to fine-tune the dynamic range of detection. The use of melting temperature differences to separate target binding from signaling moieties in the catalytic beacon method allows the use of different fluoropho...

Bioorthogonal control of metal‐ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18‐base pair recognition site of a homing endonuclease (I‐SceI), which is found by chance only once in 7×1010 bp of genomic sequences, and can thus form a near bioorthogonal pair with I‐SceI for DNAzyme activation with minimal effect on living cells. Once I‐SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg2+ to release the fluorophore‐labeled DNA fragment and produce a fluorescent turn‐on signal for Mg2+. Thus I‐SceI bioorthogonally activates the 10–23 DNAzyme for imaging of Mg2+ in HeLa cells.

A photo-regulated ATP sensor coupled with cationic DQAsomes is developed for spatiotemporally controlled imaging of ATP in the mitochondria of living cells.