Precise and dynamic imaging of extracellular pH is one crucial yet challenging task for studying cell physiological and pathological processes. Here, we construct a DNA tweezer to dynamically monitor pH changes of cellular microenvironments. The DNA tweezer contains three key elements: a three-strand ssDNA-frame labeled with cholesterol to anchor it on the cell membrane, a pH-sensitive i-motif sequence in the middle to dynamically control the switch between the "open" and "closed" states of the DNA tweezer, and a pair of FRET fluorophores (rhodamine green and rhodamine red) on the two arms of the tweezer to reflect its state. With cholesterol, a natural component of cell membranes, as an anchoring element, the sensor exhibited high cell-membrane-insertion efficiency and low cytotoxicity. Using the i-motif as a sensing element, it can quickly and reversibly respond to extracellular pH in the pH range of 5.0−7.5 and further perform real-time imaging of cell-surface-pH changes with excellent spatial and temporal resolution. Moreover, apoplastic-pH change during the alkalization process of plant roots caused by rapid-alkalinization factor (RALF1) was directly detected by the sensor, demonstrating the potential applications of the sensor in cell biology, biomedical research, and plant-tissue engineering.

DNA hydrogel is a promising biomaterial for biological and medical applications due to its native biocompatibility and biodegradability. Herein, we provide a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and hybridization between dual building blocks via their complementary TdT-polymerized DNA tails led to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated cross-linking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos (for instance, green fluorescent protein) due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by coencapsulating glucose oxidase and β-galactosidase into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye but also a promising modular platform for constructing a multiple enzyme or enzyme/DNAzyme hybrid system.

Neurotransmitters are essential chemical mediators for neuronal communication in variable neuromodulations. However, the progress of neuroscience is hampered by the shortage of suitable sensors to track neurotransmitters with high spatial and temporal resolution. Here, we introduce a self-assembled DNA-nanoprism fluorescent probe capable of nongenetically engineering the cell surface for ultrasensitive imaging of the neurotransmitter release at a single live-cell level. The DNA-nanoprism structure conjugated with three cholesterol tails enables the probe to rapidly and stably anchor on the cell surface within 10 min. The in situ detection of neurotransmitters is achieved by equipping the DNA-nanoprism with an aptamer-based “turn-on” fluorescent sensory module for the transmitter of interest. In a proof-of-concept study, we directly visualized the transient dopamine (DA) release on the cell surface with selective responsivity and high spatiotemporal precision and further explored the dynamic correlation between DA release and calcium influx triggered by high K+. This study provides a robust and sensitive tool for cell-surface-targeted imaging of neuromodulations, which might open up a new avenue to improve the understanding of neurochemistry and advance neuroscience research.

The visualization of the long noncoding RNA of prostate cancer gene 3 (lncRNA PCA3), a specific biomarker for androgen receptor-positive prostate cancer, in living cells not only directly reflects the gene expression and localization but also offers better insight into its roles in the pathological processes. Here, we loaded an entropy-driven RNA explorer (EDRE) on the TAT peptide-functionalized titanium carbide MXenes (Ti3C2–TAT) for the imaging of nuclear lncRNA PCA3 in live cells. The EDRE was condensed on the Ti3C2–TAT (Ti3C2–TAT@EDRE) by electrostatic interaction. Ti3C2–TAT@EDRE enables the entering of cells and release of TAT peptides and EDRE in the cytoplasm by the glutathione (GSH)-triggered cleavage of the disulfide bonds in Ti3C2–TAT. The released EDRE is delivered into the nucleus by the nucleus-targeted guidance of TAT peptides, and initiated by the target lncRNA PCA3, subsequently leading to the continuous accumulation of fluorescence signals. Consequently, fluorescence analysis of lncRNA PCA3 at low-picomolar concentrations in vitro as well as sensitive live cell imaging of lncRNA PCA3 in the nucleus of androgen receptor-positive LNCaP prostate cancer cells were achieved, providing a versatile strategy for the monitoring of nucleic acid biomarkers in the nucleus of living cells.

Protein phosphorylation catalyzed by protein kinase is one of the most ubiquitous and important post-translational modification and aberrant phosphorylation is closely related with a variety of human diseases, especially cancer. As the critical component of intracellular signal pathways, protein kinases are involved in regulating a number of important physiological processes, such as cell growth, metabolism, differentiation, and proliferation. Therefore, developing potent and specific analytical methods of protein kinases are of great significance for biochemical fundamental research, disease diagnosis and treatment, and drug development. In recent years, the growing number of functional nanomaterials have been exploited in the development of protein kinase biosensors. Due to their unique optical, electrical, chemical, and catalytic properties, nanomaterials play crucial roles in several key processes in bio-sensing, such as signal generation and transduction, biosensing interface fabrication, as well as biochemical signal amplification, which greatly improves the analytical performance of kinase biosensors. This paper briefly summarizes the generally used molecular recognition mechanisms of kinase detections, then gives a systematic overview of the nanomaterials-based protein kinases assays classified by the different analytical techniques. Consequently, the future development of protein kinase biosensors is prospected.