Recent Advances in DNA Nanotechnology-Based Sensing Platforms for Rapid Virus Detection.

Several major viral pandemics in history have significantly impacted the public health of human beings. The COVID-19 pandemic has further underscored the critical need for early detection and screening of infected individuals. However, current detection techniques are confronted with deficiencies in sensitivity and accuracy, restricting the capability of detecting trace amounts of viruses in human bodies and in the environments.The advent of DNA nanotechnology has opened up a feasible solution for rapid and sensitive virus determination. By harnessing the designability and addressability of DNA nanostructures, a range of rapid virus sensing platforms have been proposed. This review overviewed the recent progress, application, and prospect of DNA nanotechnology-based rapid virus detection platforms. Furthermore, the challenges and developmental prospects in this field were discussed.

DNA Walker-Driven Mass Nanotag Assembly System for Simultaneously Profiling Dual Markers of Oxidative Stress at Different Cellular Locations.

Simultaneous profiling of redox-regulated markers at different cellular sublocations is of great significance for unraveling the upstream and downstream molecular mechanisms of oxidative stress in living cells. Herein, by synchronizing dual target-triggered DNA machineries in one nanoentity, we engineered a DNA walker-driven mass nanotag (MNT) assembly system (w-MNT-AS) that can be sequentially activated by oxidative stress-associated mucin 1 (MUC1) and apurinic/apyrimidinic endonuclease 1 (APE1) from plasma membrane to cytoplasm and induce recycled assembly of MNTs for multiplex detection of the two markers by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In the working cascade, the sensing process governs the separate activation of w-MNT-AS by MUC1 and APE1 in diverse locations, while the assembly process contributes to the parallel amplification of the ion signal of the characteristic mass tags. In this manner, the differences between MCF-7, HeLa, HepG2, and L02 cells in membrane MUC1 expression and cytoplasmic APE1 activation were fully characterized. Furthermore, the oxidative stress level and dynamics caused by exogenous H2O2, doxorubicin, and simvastatin were comprehensively demonstrated by tracking the fate of the two markers across different cellular locations. The proposed w-MNT-AS coupled MS method provides an effective route to probe multiple functional molecules that lie at different locations while participating in the same cellular event, facilitating the mechanistic studies on cellular response to oxidative stress and other disease-related cellular processes.

Surface-phonon-polariton-enhanced photoinduced dipole force for nanoscale infrared imaging.

The photoinduced dipole force (PiDF) is an attractive force arising from the Coulombic interaction between the light-induced dipoles on the illuminated tip and the sample. It shows extreme sample-tip distance and refractive index dependence, which is promising for nanoscale infrared (IR) imaging of ultrathin samples. However, the existence of PiDF in the mid-IR region has not been experimentally demonstrated due to the coexistence of photoinduced thermal force (PiTF), typically one to two orders of magnitude higher than PiDF. In this study, we demonstrate that, with the assistance of surface phonon polaritons, the PiDF of c-quartz can be enhanced to surpass its PiTF, enabling a clear observation of PiDF spectra reflecting the properties of the real part of permittivity. Leveraging the detection of the PiDF of phonon polaritonic substrate, we propose a strategy to enhance the sensitivity and contrast of photoinduced force responses in transmission images, facilitating the precise differentiation of the heterogeneous distribution of ultrathin samples.

Tunable Multivalent Aptamer-Based DNA Nanostructures To Regulate Multiheteroreceptor-Mediated Tumor Recognition.

Precise mapping and regulation of cell surface receptors hold immense significance in disease treatment, such as cancer, infection, and neurodisorders, but also face enormous challenges. In this study, we designed a series of adjustable multivalent aptamer-based DNA nanostructures to precisely control their interaction with receptors in tumor cells. By profiling surface receptors on 12 cell lines using 10 different aptamers, we generated a heatmap that accurately distinguished between various tumor types based on multiple markers. We then incorporated these aptamers onto DNA origami structures to regulate receptor recognition, with patch-like structures demonstrating a tendency to be trapped on the cell surface and with tube-like structures showing a preference for internalization. Through precise control of aptamer species, valence, and geometric patterns, we found that multiheteroreceptor-mediated recognition not only favored the specific binding of nanostructures to tumor cells but also greatly enhanced intracellular uptake by promoting clathrin-dependent endocytosis. Specifically, we achieved over 5-fold uptake in different tumor cells versus normal cells using tube-like structures modified with different diheteroaptamer pairs, facilitating targeted drug delivery. Moreover, patch-like structures with triheteroaptamers guided specific interactions between macrophages and tumor cells, leading to effective immune clearance. This programmable multivalent system allows for the precise regulation of cell recognition using multiple parameters, demonstrating great potential for personalized tumor treatment.

Quality Control of Mass-Encoded Nanodevices by Compartmented DNA Origami Frames for Precision Information Coding and Logic Mapping.

Encoded nanostructures afford an ideal platform carrying multi-channel signal components for multiplexed assay and information security. However, with the demand on exclusivity and reproducibility of coding signals, precise control on the structure and composition of nanomaterials featuring fully distinguishable signals remains challenging. By using the multiplexing capability of mass spectrometry (MS) and spatial addressability of DNA origami nanostructures, we herein propose a quality control methodology for constructing mass-encoded nanodevices (namely MNTs-TDOFs) in the scaffold of compartmented tetrahedral DNA origami frames (TDOFs), in which the arrangement and stoichiometry of four types of mass nanotags (MNTs) can be finely regulated and customized to generate characteristic MS patterns. The programmability of combinatorial MNTs and orthogonality of individual compartments allows further evolution of MNTs-TDOFs to static tagging agents and dynamic nanoprobes for labeling and sensing of multiple targets. More importantly, structure control at single TDOF level ensures the constancy of prescribed MS outputs, by which a high-capacity coding system was established for secure information encryption and decryption. In addition to the multiplexed outputs in parallel, the nanodevices could also map logic circuits with interconnected complexity and logic events of c-Met recognition and dimerization on cell surface for signaling regulation by MS interrogation.

Confinement in Dual-Chain-Locked DNA Origami Nanocages Programs Marker-Responsive Delivery of CRISPR/Cas9 Ribonucleoproteins.

Delivery of CRISPR/Cas9 ribonucleoproteins (RNPs) offers a powerful tool for therapeutic genome editing. However, precise manipulation of CRISPR/Cas9 RNPs to switch the machinery on and off according to diverse disease microenvironments remains challenging. Here, we present dual-chain-locked DNA origami nanocages (DL-DONCs) that can confine Cas9 RNPs in the inner cavity for efficient cargo delivery and dual-marker-responsive genome editing in the specified pathological states. By engineering of ATP or miRNA-21-responsive dsDNAs as chain locks on the DONCs, the permeability of nanocages and accessibility of encapsulated Cas9 RNPs can be finely regulated. The resulting DL-DONCs enabled steric protection of bioactive Cas9 RNPs from premature release and deactivation during transportation while dismounting the dual chain locks in response to molecular triggers after internalization into tumor cells, facilitating the escape of Cas9 RNPs from the confinement for gene editing. Due to the dual-marker-dominated uncaging mechanism, the gene editing efficiency could be exclusively determined by the combined level of ATP and miRNA-21 in the target cellular environment. By targeting the tumor-associated PLK-1 gene, the DL-DONCs-enveloped Cas9 RNPs have demonstrated superior inhibitory effects on the proliferation of tumor cells in vitro and in vivo. The developed DL-DONCs provide a custom-made platform for the precise manipulation of Cas9 RNPs, which can be potentially applied to on-demand gene editing for classified therapy in response to arbitrary disease-associated biomolecules.

Programming the morphology of DNA origami crystals by magnesium ion strength.

Harnessing the programmable nature of DNA origami for controlling structural features in crystalline materials affords opportunities to bring crystal engineering to a remarkable level. However, the challenge of crystallizing a single type of DNA origami unit into varied structural outcomes remains, given the requirement for specific DNA designs for each targeted structure. Here, we show that crystals with distinct equilibrium phases and shapes can be realized using a single DNA origami morphology with an allosteric factor to modulate the binding coordination. As a result, origami crystals undergo phase transitions from a simple cubic lattice to a simple hexagonal (SH) lattice and eventually to a face-centered cubic (FCC) lattice. After selectively removing internal nanoparticles from DNA origami building blocks, the body-centered tetragonal and chalcopyrite lattice are derived from the SH and FCC lattices, respectively, revealing another phase transition involving crystal system conversions. The rich phase space was realized through the de novo synthesis of crystals under varying solution environments, followed by the individual characterizations of the resulting products. Such phase transitions can lead to associated transitions in the shape of the resulting products. Hexagonal prism crystals, crystals characterized by triangular facets, and twinned crystals are observed to form from SH and FCC systems, which have not previously been experimentally realized by DNA origami crystallization. These findings open a promising pathway toward accessing a rich phase space with a single type of building block and wielding other instructions as tools to develop crystalline materials with tunable properties.

Reversible Regulation of Long-Distance Charge Transport in DNA Nanowires by Dynamically Controlling Steric Conformation.

Understanding of DNA-mediated charge transport (CT) is significant for exploring circuits at the molecular scale. However, the fabrication of robust DNA wires remains challenging due to the persistence length and natural flexibility of DNA molecules. Moreover, CT regulation in DNA wires often relies on predesigned sequences, which limit their application and scalability. Here, we addressed these issues by preparing self-assembled DNA nanowires with lengths of 30-120 nm using structural DNA nanotechnology. We employed these nanowires to plug individual gold nanoparticles into a circuit and measured the transport current in nanowires with an optical imaging technique. Contrary to the reported cases with shallow or no length dependence, a fair current attenuation was observed with increasing nanowire length, which experimentally confirmed the prediction of the incoherent hopping model. We also reported a mechanism for the reversible CT regulation in DNA nanowires, which involves dynamic transitions in the steric conformation.

Enriching Reaction Intermediates in Multishell Structured Copper Catalysts for Boosted Propanol Electrosynthesis from Carbon Monoxide.

Fine-tuned catalysts that alter the diffusion kinetics of reaction intermediates is of great importance for achieving high-performance multicarbon (C2+) product generation in carbon monoxide (CO) reduction. Herein, we conduct a structural design based on Cu2O nanoparticles and present an effective strategy for enhancing propanol electrosynthesis from CO. The electrochemical characterization, operando Raman monitoring, and finite-element method simulations reveal that the multishell structured catalyst can realize the enrichment of C1 and C2 intermediates by nanoconfinement space, leading to the possibility of further coupling. Consequently, the multishell copper catalyst realizes a high Faraday efficiency of 22.22 ± 0.38% toward propanol at the current density of 50 mA cm-2.

Mass Nanotags Mediate Parallel Amplifications on Nanointerfaces for Multiplexed Profiling of RNAs.

Multiplexed profiling of RNAs aids in a comprehensive understanding of multiparameter-defined cellular processes and pathological states. We herein present a mass nanotags-enabled interfacial assembly system (MNTs-AS) with parallel amplification motors for simultaneous assaying of multiple RNAs in biosystems by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Four kinds of MNTs encoding corresponding RNA can be cyclically assembled on magnetic beads by target-triggered catalytic hairpin assembly (CHA) machineries on nanointerfaces, generating multiplexed and amplified characteristic ion signals assigned to target RNAs upon MALDI MS interrogation. By virtue of high sensitivity and multiplexing capability, the MNTs-AS-based MS assay allows precision subtyping of diverse breast cancer cells and their exosomes by multiplexed profiling of miRNA-21, miRNA-373, miRNA-155, and manganese superoxide dismutase mRNA via a single MS inquiry. This method provides a promising tool for unraveling multiple RNA-involved biological events in fundamental research and distinguishing different cancer subtypes in clinical practice.

Mass tag-encoded nanointerfaces for multiplexed mass spectrometric analysis and imaging of biomolecules.

Revealing multiple biomolecules in the physiopathological environment simultaneously is crucial in biological and biomedical research. Mass spectrometry (MS) features unique technical advantages in multiplexed and label-free analyses. However, owing to comparably low abundance and poor ionization efficiency of target biomolecules, direct MS profiling of these biological species in vitro or in situ remains a challenge. An emerging route to solve this issue is to devise mass tag (MT)-encoded nanointerfaces which specifically convert the abundance or activity of biomolecules into amplified ion signals of mass tags, offering an ideal strategy for synchronous MS assaying and mapping of multiple targets in biofluids, cells and tissues. This review provides a thorough and organized overview of recent advances in MT-encoded nanointerfaces elaborately tailored for several practical applications in multiplexed MS bioanalysis and biomedical research. First, we start with elucidation of the structural characteristics and working principle of MT-encoded nanointerfaces in specific labeling and sensing of multiple biological targets. In addition, we further discuss the application scenarios of MT-encoded nanointerfaces particularly in multiplexed biomarker assays, cell analysis, and tissue imaging. Finally, the current challenges are pointed out and future prospects of these nanointerfaces in MS analysis are forecast.

A Telomerase-Assisted Strategy for Regeneration of DNA Nanomachines in Living Cells.

DNA nanomachines have been engineered into diverse personalized devices for diagnostic imaging of biomarkers; however, the regeneration of DNA nanomachines in living cells remains challenging. Here, we report an ingenious DNA nanomachine that can implement telomerase (TE)-activated regeneration in living cells. Upon apurinic/apyrimidinic endonuclease 1 (APE1)-responsive initiation of the nanomachine, the walker of the nanomachine moves along tracks regenerated by TE, generating multiply amplified signals through which APE1 can be imaged in situ. Additionally, augmentation of the signal due to the regeneration of the nanomachines could reveal differential expression of TE in different cell lines. To the best of our knowledge, this is the first proof-of-concept demonstration of the use of biomarkers to assist in the regeneration of nanomachines in living cells. This study offers a new paradigm for the development of more applicable and efficient DNA nanomachines.

Lipid droplet-hitchhiking probe creates Trojan foam cells for fluorescence/photoacoustic imaging of atherosclerotic plaques.

Since atherosclerosis, a disease characterized by abnormal arterial lipid deposition, may lead to fatal cardiovascular diseases, imaging of atherosclerotic plaques is of great value for their pathological assessment. In this study, we propose a lipid droplet (LD)-hitchhiking strategy to in situ create Trojan foam cells for fluorescence/photoacoustic imaging of atherosclerotic plaques via homologous targeting effect. In our design, functional liposomes (DCP liposomes) composed of phospholipid dioleoylphosphatidylserine (DOPS), a novel LD inducer we found, and Cypate-PC, a synthesized lipid-like molecular probe, have demonstrated great capability of inducing LDs in monocytes/macrophages while being enveloped into the resulting Trojan foam cells. Taking advantage of homologous targeting effect, the imaging probe hitchhikes on the LDs in Trojan foam cells for targeted transport to the plaque sites. Moreover, the confinement in highly hydrophobic LDs endows the imaging probe with high efficiency in light absorption, enabling greatly intensified fluorescence/photoacoustic signals. The DCP liposomes have shown great potency in inducing the generation of Trojan foam cells, and eventually ex vivo fluorescence imaging and in vivo photoacoustic imaging of atherosclerotic plaques. The proposed strategy provides more insights into the design of targeted imaging methodologies, and also an effective avenue to facilitate the evaluation and subsequent treatment of atherosclerotic plaques.

Insights into electrochemiluminescence dynamics by synchronizing real-time electrical, luminescence, and mass spectrometric measurements.

Electrochemiluminescence (ECL) comprises a sophisticated cascade of reactions. Despite advances in mechanistic studies by electrochemistry and spectroscopy, a lack of access to dynamic molecular information renders many plausible ECL pathways unclear or unproven. Here we describe the construction of a real-time ECL mass spectrometry (MS) platform (RT-Triplex) for synchronization of dynamic electrical, luminescent, and mass spectrometric outputs during ECL events. This platform allows immediate and continuous sampling of newly born species at the Pt wire electrode of a capillary electrochemical (EC) microreactor into MS, enabling characterization of short-lived intermediates and the multi-step EC processes. Two ECL pathways of luminol are validated by observing the key intermediates α-hydroxy hydroperoxide and diazaquinone and unraveling their correlation with applied voltage and ECL emission. Moreover, a "catalytic ECL route" of boron dipyrromethene (BODIPY) involving homogeneous oxidation of tri-n-propylamine with the BODIPY radical cation is proposed and verified.

Top-Down Rational Engineering of Heteroatom-Doped Graphene Quantum Dots for Laser Desorption/Ionization Mass Spectrometry Detection and Imaging of Small Biomolecules.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is widely applied in mapping macrobiomolecules in tissues, but it is still limited in profiling low-molecular-weight (MW) compounds (typically metabolites) due to ion interference and suppression by organic matrices. Here, we present a versatile "top-down" strategy for rational engineering of carbon material-based matrices, by which heteroatom-doped graphene quantum dots (HGQDs) were manufactured for LDI MS detection and imaging of small biomolecules. The HGQDs derived from parent materials inherited the π-conjugated networks and doping sites for promoting energy transfer and negative ion generation, while their extremely small size guaranteed the matrix uniformity and signal reproducibility in LDI MSI. Compared to other HGQDs, nitrogen-doped graphene quantum dots (NGQDs) exhibited superior capability of assisting LDI of various small molecules, including amino acids, fatty acids, saccharides, small peptides, nucleobases, anticancer drugs, and bisphenol pollutants. Density functional theory simulations also corroborated that the LDI efficiency was markedly raised by the proton-capturing pyridinic nitrogen species and compromised by the electron-deficient boron dopants. NGQDs-assisted LDI MS further enabled label-free investigation on enzyme kinetics using an ordinary short peptide as the substrate. Moreover, due to the high salt tolerance and signal reproducibility, the proposed negative-ion NGQDs-assisted LDI MSI was able to reveal the abundance and distribution of low-MW species in rat brain tissue and achieved the imaging of low-MW lipids in coronally sectioned rat brains subjected to traumatic brain injury. Our work offers a new route for customizing nanomaterial matrices toward LDI MSI of small biomolecules in biomedical and pathological research.

In situ formed N-containing copper nanoparticles: a high-performance catalyst toward carbon monoxide electroreduction to multicarbon products with high faradaic efficiency and current density.

Developing efficient catalysts for electrochemical carbon monoxide reduction (ECOR) with high faradaic efficiency (FE) and current density is highly desirable. In this work, we demonstrate that the N-containing Cu nanoparticles formed in situ by the reconstruction of cuprous 7,7,8,8-tetracyanoquinodimethane possess high-performance ECOR ability. Impressively, the N-containing Cu nanoparticle catalyst presented the highest FE of 81.31% towards multicarbon products with a high commercial-grade partial current density of 162.62 mA cm-2, which is among the best of the reported Cu-based ECOR catalysts at -0.69 V versus the reversible hydrogen electrode. The retained ligand on the formed catalyst via the convenient in situ formation is crucial for the selectivity of multicarbon products. This work will arouse enthusiasm for the utilization of reconstruction features for designing ligand-containing catalysts with enhanced artificial carbon fixation ability.

A six-plex switchable DNA origami cipher disk for tandem-in-time cryptography.

We report a DNA origami cipher disk (DOCD) allowing random, continuous and reversible switchover between six visibly different patterns in response to the input DNA strands. A DOCD-enabled tandem-in-time cryptographic protocol was thereby established by using a string of DNA strands as a carrier for accurate information encoding and transmission.

Two-Stage Assembly of Nanoparticle Superlattices with Multiscale Organization.

Self-assembly processes, while promising for enabling the fabrication of complexly organized nanomaterials from nanoparticles, are often limited in creating structures with multiscale order. These limitations are due to difficulties in practically realizing the assembly processes required to achieve such complex organizations. For a long time, a hierarchical assembly attracted interest as a potentially powerful approach. However, due to the experimental limitations, intermediate-level structures are often heterogeneous in composition and structure, which significantly impacts the formation of large-scale organizations. Here, we introduce a two-stage assembly strategy: DNA origami frames scaffold a coordination of nanoparticles into designed 3D nanoclusters, and then these clusters are assembled into ordered lattices whose types are determined by the clusters' valence. Through modulating the nanocluster architectures and intercluster bindings, we demonstrate the successful formation of complexly organized nanoparticle crystals. The presented two-stage assembly method provides a powerful fabrication strategy for creating nanoparticle superlattices with prescribed unit cells.

Multistage Photoactivatable Zinc-Responsive Nanodevices for Monitoring and Regulating Dysfunctional Islet ß-Cells.

The dysfunctional islet ß-cell triggered by excessive deposition of Zn2+ constituted a striking indicator of the occurrence of diabetic disease. However, it remained a formidable challenge to reflect the real-time function of ß-cell by monitoring the Zn2+ content. Herein, multistage photoactivatable Zn2+-responsive nanodevice (denoted as AD2@USD1) was presented for sensing, regulating, and evaluating Zn2+ levels in dysfunctional islet ß-cells. The photoactivated signatures on the satellite shell layer of the nanodevices and the internally loaded chelating factors effectively identified and intervened in the real-time concentration of Zn2+, the photothermal feedback component decorated on the inner core permitted the assessment of the post-intervention Zn2+ levels, achieving an integrated intervention and prognostic assessment in response to the abnormal islet ß-cell function induced by Zn2+ deposition. In this way, one strategy for sensing and regulating islet ß-cell function-oriented to Zn2+ was established. Our study introduced AD2@USD1 as a tool for effectively sensing, adjusting, and assessing the Zn2+ level in islet ß-cells with abnormalities, gaining a potential breakthrough in the treatment of diabetes.

Sense and Validate: Fluorophore/Mass Dual-Encoded Nanoprobes for Fluorescence Imaging and MS Quantification of Intracellular Multiple MicroRNAs.

Simultaneously monitoring and quantifying intracellular multiple microRNAs (miRNAs) is highly essential to clinical diagnosis and pathological research. However, revealing the intracellular distribution of multiple miRNAs while determining their content in a multiplex and quantitative format remains challenging. Considering the respective technical merit of fluorescence imaging and mass spectrometry (MS) in in situ detection and multiplex assaying, we herein propose fluorophore/mass dual-encoded nanoprobes (FMNPs) that can execute target-triggered hairpin self-assembly to enable in situ amplified imaging and follow-up MS quantification of intracellular multiple miRNAs. The FMNPs responsive to the target miRNA were constructed by codecorating gold nanoparticles (AuNPs) with locked hairpin DNA probes (LH1) and corresponding mass tags (MTs) for fluorescent and mass spectrometric dual-modal readout. Cellular miRNAs can separately trigger recycled hairpin self-assembly, leading to the continuous liberation of fluorophore-labeled bolt DNA (bDNA) for fluorescence imaging in cells. Moreover, the postreaction FMNPs afford an extra chance to validate the fluorescence output of miRNA-21 and miRNA-141 by accurate MS quantification relying on the ion signal of the barcoded MTs. Fluorescence imaging and MS quantification of miRNA-21 and miRNA-141 have also been successfully accomplished in different cell lines, highlighting its potential in cell subtyping. This "sense-and-validate" strategy creates a new modality for assaying multiple intracellular miRNAs and holds great promise in unveiling multicomponent-involved events in cellular processes and determining multiple biomarkers in accurate clinical diagnosis.