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DNA hydrogels are biocompatible and are suitable for many biomedical applications. However, to be useful imaging probes or drug carriers, the ordinary bulk size of DNA hydrogels must be overcome. Here we put forward a new strategy for fabricating a novel and simple protein-scaffolded DNA nanohydrogel, constructed through a direct DNA self-assembly using three types of streptavidin (SA)-based DNA tetrad for the activation of imaging and targeting therapy of cancer cells. The DNA nanohydrogels are easily prepared, and we show that by varying the initial concentration of DNA tetrad, it is possible to finely control their size within nanoscale range, which are favorable as carriers for intracellular imaging and transport. By further incorporating therapeutic agents and tumor-targeting MUC1 aptamer, these multifunctionalized SA-scaffolded DNA nanohydrogels (SDH) can specifically target cancer cells and selectively release the preloaded therapeutic agents via a structure switching when in an ATP-rich intracellular environment, leading to the activation of the fluorescence and efficient treatment of cancer cells. With the advantages of facile modular design and assembly, effective cellular uptake, and excellent biocompatibility, the method reported here has the potential for the development of new tunable DNA nanohydrogels with multiple synergistic functionalities for biological and biomedical applications.

DNA molecular probes have emerged as a powerful tool for RNA imaging. Hurdles in cell-specific delivery and other issues such as insufficient stability, limited sensitivity, or slow reaction kinetics, however, hinder the further application of DNA molecular probes in vivo. Herein, we report an aptamer-tethered DNA polymer for cell-specific transportation and amplified imaging of RNA in vivo via a DNA cascade reaction. DNA polymers are constructed through an initiator-triggered hybridization chain reaction using two functional DNA monomers. The prepared DNA polymers show low cytotoxicity and good stability against nuclease degradation and enable cell-specific transportation of DNA circuits via aptamer-receptor binding. Moreover, assembling the reactants of hairpins C1 and C2 on the DNA polymers accelerates the response kinetics and improves the sensitivity of the cascade reaction. We also show that the DNA polymers enable efficient imaging of microRNA-21 in live cells and in vivo via intravenous injection. The DNA polymers provide a valuable platform for targeted and amplified RNA imaging in vivo, which holds great implications for early clinical diagnosis and therapy.

Microfluidic paper analytical devices (μPADs) are smart and accessible substituents to traditional counterparts in point-of-care tests (POCT), which exploited delicate control over passive fluid in microscale for rich functions. In this work, we are extending such control by introducing novel ways to generate 1D and 2D gradients on μPADs. It is achieved by using paper-capillary-based serial sampling. The paper capillary is composed of a concaved paper channel sealed with tape, with test pads properly distributed aside. In such a structure, the liquid can not only quickly and automatically flow along the capillary but also be continuously consumed by the peripheral test pads. Thus, when we do serial sampling, an abnormal liquid chasing effect can be observed, where the later liquid sample chases and surpasses the earlier parts in the paper capillary, leading to reversely ordered sample distribution compared with that in a typical glass capillary. This specialty allows for fast, ordered, and tunable sequential sampling and enables efficient generation of 1D and 2D concentration gradient with one, two, and even three components on μPADs. Besides, we verified the applicability of this technique for arrayed assays, including 1D serial dilution-based metal ion colorimetry and a 2D bacterial antibiotic susceptibility test for synergic effect evaluations, which paves the way for high-throughput sample analysis and information-rich condition screening on μPADs.

Hybridization chain reaction (HCR) circuits are valuable approaches to monitor low-abundance mRNA, and current HCR is still subjected to issues such as limited amplification efficiency, compromised localization resolution, or complicated designs. We report a novel branched HCR (bHCR) circuit for efficient signal-amplified imaging of mRNA in living cells. The bHCR can be realized using a simplified design by hierarchically coupling two HCR circuits with two split initiator fragments of the secondary HCR circuit incorporated in the probes for the primary HCR circuit. The bHCR circuit enables one to generate a hyperbranched assembly seeded from a single target initiator, affording the potential for localizing single target molecules in live cells. In vitro assays show that bHCR offers very high amplification efficiency and specificity in single mismatch discrimination with a detection limit of 500 fM. Live cell studies reveal that bHCR displays intense fluorescence spots indicating mRNA localization in living cells with improved contrast. The bHCR method can provide a useful platform for low-abundance biomarker detection and imaging for cell biology and diagnostics.

Design of DNA self-assembly with reversible responsiveness to external stimuli is of great interest for diverse applications. We for the first time develop a pH-responsive, fully reversible hybridization chain reaction (HCR) assembly that allows sensitive sensing and imaging of pH in living cells. Our design relies on the triplex forming sequences that form DNA triplex with toehold regions under acidic conditions and then induce a cascade of strand displacement and DNA assembly. The HCR assembly has shown dynamic responses in physiological pH ranges with excellent reversibility and demonstrated the potential for in vitro detection and live-cell imaging of pH. Moreover, this method affords HCR assemblies with highly localized fluorescence responses, offering advantages of improving sensitivity and better selectivity. The proton-fueled, reversible HCR assembly may provide a useful approach for pH-related cell biology study and disease diagnostics.

The efficient detection and in situ monitoring of telomerase activity is of great importance for cancer diagnosis and biomedical research. Here we report for the first time that the development of a novel multivalent self-assembled DNA polymer, constructed through telomerase primer sequence (ITS) triggered hybridization chain assembly using two functional hairpin probes (tumor-trageting aptamer modified H1 and signal probe modified H2), for sensitive detection and imaging of telomerase activity in living cells. After internalizing into the tumor cells by multivalent aptamer targeting, the ITS on DNA polymers can be elongated by intracellular telomerase to generate telomere repeat sequences that are complementary with the signal probe, which can proceed along the DNA polymers, and gradually light up the whole DNA polymers, leading to an enhanced fluorescence signal directly correlated with the activity of telomerase. Our results demonstrated that the developed DNA polymer show excellent performance for specifically detecting telomerase activity in cancer cells, dynamically monitoring the activity change of telomerase in response to telomerase-based drugs, and efficiently distinguishing cancer cells from normal cells. The proposed strategy may afford a valuable tool for the monitoring of telomerase activity in living cells and have great implications for biological and diagnostic applications.

DNA nanodevices that mimic natural biomolecular machines changing configurations in response to external inputs have enabled smart sensors to live cell imaging. We report for the first time the development of a dynamic DNA nanomachine that is anchored on a cell’s surface and undergoes pH-responsive triplex–duplex conformation switching, allowing tunable sensing and imaging of extracellular pH. Results reveal that the DNA nanomachine can be stably anchored on the cell surface via multiple anchors, and the adjustment of C+G-C content in the switch element confers tunability of pH response windows. The anchored DNA nanomachine also demonstrates desirable sensitivity, excellent reversibility, and quantitative ability for extracellular pH detection and imaging. This cell surface-anchored pH-responsive DNA nanomachine can provide a useful platform for pH sensing in extracellular microenvironments and diagnostics of different pH-related diseases.

Unique physicochemical characteristics of graphitic carbon nitride (g-CN) nanosheets suit them to be a useful tool for two-photon fluorescence bioimaging. Current g-CN nanosheets based imaging probes typically use the "always-on" design strategies, which may suffer from increased fluorescence background and limited contrast. To advance corresponding applications, g-CN nanosheets based activatable two-photon fluorescence probes remain to be explored. For the first time, we developed an activatable two-photon fluorescence probe, constructed from a nanoassembly of g-CN nanosheets and hyaluronic acid (HA)-gold nanoparticles (HA-AuNPs), for detection and imaging of hyaluronidase (HAase) in cancer cells. The deliberately introduced HA in our design not only functions as the buffering layer for stabilizing AuNPs and inducing corresponding self-assembly on g-CN nanosheets but also as a pilot for targeting HA receptors overexpressed on cancer cell surfaces. Our results show that the developed nanoassembly enables specific detection and activatable imaging of HAase in cancer cells and deep tissues, with superb signal-to-background ratio and high sensitivity. This nanoassembly can afford a promising platform for highly specific and sensitive imaging of HAase and for related cancer diagnosis.

Graphitic C3N4 (g-C3N4) nanosheets are a type of emerging graphene-like carbon-based nanomaterials with high fluorescence and large specific surface areas that hold great potential for biosensor applications. However, current g-C3N4 based biosensors have prevailingly been limited to coordination with metal ions, and it is of great significance to develop new designs for g-C3N4 nanosheets based biosensors toward biomarkers of general interest. We report the development of a novel g-C3N4 nanosheet-based nanosensor strategy for highly sensitive, single-step and label-free detection of tyrosinase (TYR) activity and its inhibitor. This strategy relies on the catalytic oxidation of tyrosine by TYR into melanin-like polymers, which form a nanoassembly on the g-C3N4 nanosheets and quench their fluorescence. This strategy was demonstrated to provide excellent selectivity and superior sensitivity and to enable rapid screening for TYR inhibitors. Therefore, the developed approach might create a useful platform for diagnostics and drugs screening for TYR-based diseases including melanoma cancer.