Ionic Strength-Mediated "DNA Corona Defects" for Efficient Arrangement of Single-Walled Carbon Nanotubes.

Single-stranded DNA oligonucleotides wrapping on the surface of single-walled carbon nanotubes (SWCNTs), described as DNA corona, are often used as a dispersing agent for SWCNTs. The uneven distribution of DNA corona along SWCNTs is related to the photoelectric properties and the surface activity of SWCNTs. An ionic strength-mediated "DNA corona defects" (DCDs) strategy is proposed to acquire an exposed surface of SWCNTs (accessible surface) as large as possible while maintaining good dispersibility via modulating the conformation of DNA corona. By adjusting the solution ionic strength, the DNA corona phase transitioned from an even-distributed and loose conformation to a locally compact conformation. The resulting enlarged exposed surface of SWCNTs is called DCDs, which provide active sites for molecular adsorption. This strategy is applied for the arrangement of SWCNTs on DNA origami. SWCNTs with ≈11 nm DCD, providing enough space for the adsorption of "capture ssDNA" (≈7 nm width required for 24-nt) extended from DNA origami structures are fabricated. The DCD strategy has potential applications in SWCNT-based optoelectronic devices.

The controllable patterning of tannic acid on DNA origami.

Tannic acid-based patterning is crucial for its applications in bioengineering, including multifunctional coatings, biosensors, and biochips. However, tannic acid (TA) patterning is challenging owing to the rapid polymerization kinetics of tannins and their strong adhesion towards most surfaces or objects. Herein, we report a strategy for controllable TA nanopatterning based on DNA origami templates. Protruding clustered ssDNA (pcDNA) from DNA origami tiles served as indexes for the selective deposition of TA due to the high flexibility of ssDNA and exposed aromatic bases, which provide active sites for TA-DNA interactions. Next, by exploiting the pH-sensitive degradation of TA polymers, controllable 'erasing' and 'rewriting' of TA nanopatterns were performed. Finally, combining the high adhesion and selective deposition, the TA polymers as a glue modified on the edges of origami tiles directed the reversible association/disassociation of origami multimers. Our strategy provides a simple approach for the controllable nanopatterning of TA, enabling its unique properties to tailor surface patterns for applications in materials science and biomedicine.

Highly Uniform DNA Monolayers Generated by Freezing-Directed Assembly on Gold Surfaces Enable Robust Electrochemical Sensing in Whole Blood.

Assembling DNA on solid surfaces is fundamental to surface-based DNA technology. However, precise control over DNA conformation and organization at solid-liquid interfaces remains a challenge, resulting in limited stability and sensitivity in biosensing applications. We herein communicate a simple and robust method for creating highly uniform DNA monolayers on gold surfaces by a freeze-thawing process. Using Raman spectroscopy, fluorescent imaging, and square wave voltammetry, we demonstrate that thiolated DNA is concentrated and immobilized on gold surfaces with an upright conformation. Moreover, our results reveal that the freezing-induced DNA surfaces are more uniform, leading to improved DNA stability and target recognition. Lastly, we demonstrate the successful detection of a model drug in undiluted whole blood while mitigating the effects of biofouling. Our work not only provides a simple approach to tailor the DNA-gold surface for biosensors but also sheds light on the unique behavior of DNA oligonucleotides upon freezing on the liquid-solid interface.

Functions and applications of artificial intelligence in droplet microfluidics.

Droplet microfluidics has emerged as a powerful technology to perform high-throughput experiments, while artificial intelligence (AI) serves as a functional tool to analyze a large set of multiplex data. Their convergence creates new opportunities in autonomous system optimization and control, enabling various innovative functions and applications. In this study, we elucidate the basic principles of AI and elaborate on its main functions. The intelligent microfluidic systems applied in droplet generation, material synthesis, and biological analysis are summarized, with their working mechanisms and enabled new functions highlighted. Moreover, we elucidate current challenges in a more widespread combination of AI and droplet microfluidics and offer our perspectives on potential strategies to tackle these challenges. We hope that this review can deepen our understanding of intelligent droplet microfluidics and inspire more functional designs tailored to emerging demands.

Microwell array chip-based single-cell analysis.

Single-cell profiling is key to uncover the cellular heterogeneity and drives deep understanding of cell fate. In recent years, microfluidics has become an ideal tool for single-cell profiling owing to its benefits of high throughput and automation. Among various microfluidic platforms, microwell has the advantages of simple operation and easy integration with in situ analysis ability, making it an ideal technique for single-cell studies. Herein, recent advances of single-cell analysis based on microwell array chips are summarized. We first introduce the design and preparation of different microwell chips. Then microwell-based cell capture and lysis strategies are discussed. We finally focus on advanced microwell-based analysis of single-cell proteins, nucleic acids, and metabolites. The challenges and opportunities for the development of microwell-based single-cell analysis are also presented.

Hierarchical DNA branch assembly-encoded fluorescent nanoladders for single-cell transcripts imaging.

Spatial visualization of single-cell transcripts is limited by signal specificity and multiplexing. Here, we report hierarchical DNA branch assembly-encoded fluorescent nanoladders, which achieve denoised and highly multiplexed signal amplification for single-molecule transcript imaging. This method first offers independent RNA-primed rolling circle amplification without nonspecific amplification based on circular DNAzyme. It then executes programmable DNA branch assembly on these amplicons to encode virtual signals for visualizing numbers of targets by FISH. In theory, more virtual signals can be encoded via the increase of detection spectral channels and repeats of the same sequences on barcode. Our method almost eliminates nonspecific amplification in fixed cells (reducing nonspecific spots of single cells from 16 to nearly zero), and achieves simultaneous quantitation of nine transcripts by using only two detection spectral channels. We demonstrate accurate RNA profiling in different cancer cells, and reveal diverse localization patterns for spatial regulation of transcripts.

Branched immunochip-integrated pairwise barcoding amplification exploring the spatial proximity of two post-translational modifications in distinct cell subpopulations.

Investigating the spatial information of post-translational modifications (PTMs) in distinct cell subpopulations represents a new direction toward single-cell analysis. The specific capture of cell populations combined with PTM spatial proximity visualization making it practically challenging. Here, we develop branched immunochip-integrated pairwise barcoding amplification, termed biChip-PBA, which can perform the respective capture of cell subpopulations expressing different membrane proteins and successive PBA-based fluorescence imaging of PTM proximities. Our work may provide multilevel information for new insights into epigenetic regulation and cell function.

Lighting Up Nucleic Acid Modifications in Single Cells with DNA-Encoded Amplification.

Nucleic acids are naturally decorated with various chemical modifications at nucleobases. Most nucleic acid modifications (NAMs) do not alter Watson-Crick base pairing but can regulate gene expression known as "epigenetics". Their abundances present a very wide range, approximately 10-2 to 10-6 of total bases. Different NAMs may coexist in spatial proximity (e.g., <20 nm) in the crowded intracellular environment. Considering the highly dynamic chromatin accessibility (physical access to DNA), the NAMs in inaccessible DNA probably plays different roles. These multilayered features of NAMs vary from cell to cell. Our understanding of the function and mechanism of NAMs in biological processes and disease states has largely been driven by the expanding array of sequencing-based methodologies. However, an underexplored aspect is the measurement of the subcellular distribution, spatial proximity, and inaccessibility of NAMs in single cells. In recent years, we have developed new approaches that light up single-cell NAMs with single-site sensitivity. These methods are mainly based on the integration of chemical or chemoenzymatic tools, DNA amplification and nanotechnology, and/or microfluidics. An overview of these methods together with conventional methods such as immunofluorescence (IF) and fluorescence in situ hybridization (FISH) is provided in this Account.Our laboratory has proposed DNA-encoded amplification (DEA) as the main strategy for developing a set of single-cell NAM imaging methods. In brief, DEA transforms the different features of NAMs into unique DNA primers for rolling circle amplification (RCA) followed by FISH imaging. The first method is base-encoded amplifying FISH (BEA-FISH), in which we convert individual NAMs into RCA primers via chemoselective labeling and click bioconjugation. It enables the in situ visualization of low-abundance NAMs (e.g., 5hmU), which is impracticable by conventional methods. We subsequently developed pairwise proximity-differentiated amplifying FISH (PPDA-FISH), which integrates BEA-FISH with DNA nanotechnology. PPDA-FISH utilizes proximity ligation and toehold strand displacement to label the adjacent site of two different NAMs (one-to-one proximity) and their respective residual sites with three unique RCA probes. It achieves simultaneous counting of the above-mentioned three types of modified sites in the same cells. The third method is cellular macromolecule-tethered DNA walking indexing (Cell-TALKING) to probe more than two NAMs within the same nanoenvironments. Cell-TALKING uses dynamic DNA proximity cleavage to continuously activate different preblocked RCA primers (for each NAM) near one walking probe (for one target molecule). We have explored three NAMs around one histone (one-to-many proximity) in different cancer cell lines and clinical specimens. Then, we describe a single-cell hydrogel encoding amplification (scHEA) method by integrating droplet microfluidics with BEA-FISH. This method generates hydrogel beads that encapsulate single cells and their genomic DNA after cell lysis. It realizes the analysis of global (accessible and inaccessible) DNA from the same cells. We find that the global levels of both 5hmC and 5hmU in single cells can distinguish different breast cancer cells. Finally, the current limitations of these strategies and the future development directions are also discussed. We hope that this Account can spark new ideas and invite new efforts from different disciplines for single-cell NAM analysis.

Surface Engineering of Lipid Vesicles Based on DNA Nanotechnology.

Lipid vesicle research is of great significance in the field of biomedicine and great progress has been made in recent years, in which the surface engineering on lipid membranes plays an important role. By introducing new active sites on membrane surface, the physicochemical properties of vesicles are regulated and the biological functions are extended. DNA nanotechnology is an excellent tool for surface engineering of vesicles and has attracted more and more attention. In this Review, the interaction between DNA and lipid membrane is presented. Subsequently, recent advances in the applications of vesicle-surface-engineering based on DNA nanotechnology are highlighted. DNA nanostructures are used to mimic membrane proteins in the system of artificial liposome vesicles. Surface-engineered extracellular vesicles (EVs) based on DNA nanotechnology are applied to achieve non-invasive early screening of diseases with high sensitivity and precision. Finally, challenges and prospects for future development in this field are discussed.

Universal Exponential Amplification Confers Multilocus Detection of Mutation-Prone Virus.

The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has spread rapidly around the world. Accurate and scalable diagnostics are essential for immediate intervention and control of viral transmission. Currently reported diagnostics are rapid and sensitive, yet most are limited by their principle of single-locus identification and suffer from false-negative results because of the mutation-prone nature of RNA viruses. Here, we propose a multilocus detection method for SARS-CoV-2 based on a modified loop-mediated isothermal amplification with a pair of universal primers. The sequence-specific probes are designed to recognize the sequence of nucleocapsid protein (N) and the open reading frame 1ab (Orf1ab) gene from the SARS-CoV-2 genome. In the presence of a target locus, separated probes are ligated to be an intact template, the bipartite ends of which are repetitive sequences for the sequential binding of universal primers to initiate strand displacement. A kind of flap structure-dependent endonuclease is involved in cleaving multicolor TaqMan probes during multiplex amplification, realizing a real-time and multiplex analysis. We evaluated the quantitative performance of the developed method with spiked samples using synthetic target RNA, resulting in a limit of detection as low as 250 aM. Furthermore, the feasibility of multilocus detection was validated using various mutation-prone genes, demonstrating a significant potential for accurate analysis of SARS-CoV-2 and holding great promise for the clinical diagnosis of other infectious diseases.

A bifunctional chemical signature enabling RNA 4-thiouridine enrichment sequencing with single-base resolution.

Both sequence enrichment and base resolution are essential for accurate sequencing analysis of low-abundance RNA. Yet they are hindered by the lack of molecular tools. Here we report a bifunctional chemical signature for RNA 4-thiouridine (4sU) enrichment sequencing with single-base resolution. This chemical signature is designed for specific 4sU labeling with two functional parts. One part is a distal alkynyl group for the biotin-assisted pulldown enrichment of target molecules via click chemistry crosslinking. The other part is a -NH group proximal to the pyrimidine ring of 4sU. It allows 4sU-to-cytosine transition during the polymerase-catalyzed extension reaction based on altering hydrogen-bonding patterns. Ultimately, the 4sU-containing RNA molecules can be enriched and accurately analyzed by single-base resolution sequencing. The proposed method also holds great potential to investigate transcriptome dynamics integrated with high-throughput sequencing.

Addition-Elimination Mechanism-Activated Nucleotide Transition Sequencing for RNA Dynamics Profiling.

Dynamic information of intracellular transcripts is essential to understand their functional roles. Routine RNA-sequencing (RNA-seq) methods only measure RNA species at a steady state and do not provide RNA dynamic information. Here, we develop addition-elimination mechanism-activated nucleotide transition sequencing (AENT-seq) for transcriptome-wide profiling of RNA dynamics. In AENT-seq, nascent transcripts are metabolically labeled with 4-thiouridine (4sU). The total RNA is treated with N2H4·H2O under aqueous conditions. N2H4·H2O is demonstrated to convert 4sU to 4-hydrazino cytosine (C*) based on an addition-elimination chemistry. C* is regarded as cytosine (C) during the DNA extension process. This 4sU-to-C transition marks nascent transcripts, so it enables sequencing analysis of RNA dynamics. We apply our AENT-seq to investigate transcript dynamic information of several genes involved in cancer progression and metastasis. This method uses a simple chemical reaction in aqueous solutions and will be rapidly disseminated with extensive applications.

Development of pH-responsive nanocomposites with remarkably synergistic antibiofilm activities based on ultrasmall silver nanoparticles in combination with aminoglycoside antibiotics.

Bacterial biofilms are responsible for many chronic infections because antibacterial agents exhibit poor penetration into the dense matrix barrier and cannot easily reach the internal bacteria. Herein, we reported pH-responsive nanocomposites (PDA@Kana-AgNPs) that could penetrate and disperse biofilms, which were synthesized by the combination of ultrasmall silver nanoparticles (AgNPs) and kanamycin, and then coating with polydopamine. Confocal fluorescence imaging indicated that PDA@Kana-AgNPs could respond to the acidic microenvironment of biofilms, leading to biofilm-triggered on- demand drug release in situ. The zone of inhibition test and Resazurin assay showed that the combination of kanamycin and AgNPs had greater antimicrobial activity against test strains (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Escherichia coli BL21) than when applied separately. The crystal violet staining test demonstrated that biofilms were effectively dispersed by the proposed nanocomposites. Biocompatibility was also evaluated, which showed that PDA@Kana-AgNPs were non-toxic to mammalian cells. Therefore, the proposed pH-responsive nanocomposites held great potential for efficient antibiotics delivery and showed synergistic antibacterial and antibiofilm activities. This strategy could also be used to encapsulate a variety of antibiotics in combination with other drugs or materials, thereby showing therapeutic potential in preventing biofilm-related infections and realizing fluorescence imaging in situ.

Construction of a Mesoporous Ceria Hollow Sphere/Enzyme Nanoreactor for Enhanced Cascade Catalytic Antibacterial Therapy.

Nanozyme has been regarded as one of the antibacterial agents to kill bacteria via a Fenton-like reaction in the presence of H2O2. However, it still suffers drawbacks such as insufficient catalytic activity in near-neutral conditions and the requirement of high H2O2 levels, which would minimize the side effects to healthy tissues. Herein, a mesoporous ceria hollow sphere/enzyme nanoreactor is constructed by loading glucose oxidase in the mesoporous ceria hollow sphere nanozyme. Due to the mesoporous framework, large internal voids, and high specific surface area, the obtained nanoreactor can effectively convert the nontoxic glucose into highly toxic hydroxyl radicals via a cascade catalytic reaction. Moreover, the generated glucose acid can decrease the localized pH value, further boosting the peroxidase-like catalytic performance of mesoporous ceria. The generated hydroxyl radicals could damage severely the cell structure of the bacteria and prevent biofilm formation. Moreover, the in vivo experiments demonstrate that the nanoreactor can efficiently eliminate 99.9% of bacteria in the wound tissues and prevent persistent inflammation without damage to normal tissues in mice. This work provides a rational design of a nanoreactor with enhanced catalytic activity, which can covert glucose to hydroxyl radicals and exhibits potential applications in antibacterial therapy.

Bioorthogonal Chemical Signature Enabling Amplified Visualization of Cellular Oxidative Thymines.

Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.

Controllable synthesis of iron-polyphenol colloidal nanoparticles with composition-dependent photothermal performance.

Iron-polyphenol nanoparticles are usually prepared with nontoxic plant polyphenols as a main building block, which are an emerging photothermal agent for photothermal therapy. However, till now, few works have been made on the controllable synthesis of iron-polyphenol nanoparticles with tunable composition, as well as investigation of the relationship between material composition and photothermal property. In the present study, iron-polyphenol colloidal nanoparticles with tunable diameter (21-303 nm) and ion content (9.2-97.6 mg/g), as well as high colloidal stability are successfully synthesized using different polyphenols (such as tannic acid, epigallocatechin gallate, gallic acid, epicatechin and proanthocyanidin) as a ligand. In addition, photothermal performance is highly dependent on the organic ligand, iron content and particle size. Higher iron content and smaller diameter can contribute to higher photothermal performance. The iron-polyphenol nanoparticles with the optimal iron content and particle size are selected as a photothermal agent. They can effectively inhibit the tumour growth in vivo. The current work demonstrates a general synthesis strategy for iron-polyphenol colloidal nanoparticles with tailorable composition and clarifies the relationship between material composition and photothermal performance. Moreover, it is conductive to the rational design of polyphenol-based photothermal agents for theranostic applications.

Cellular macromolecules-tethered DNA walking indexing to explore nanoenvironments of chromatin modifications.

Exploring spatial organization and relationship of diverse biomolecules within cellular nanoenvironments is important to elucidate the fundamental processes of life. However, it remains methodologically challenging. Herein, we report a molecular recognition mechanism cellular macromolecules-tethered DNA walking indexing (Cell-TALKING) to probe the nanoenvironments containing diverse chromatin modifications. As an example, we characterize the nanoenvironments of three DNA modifications around one histone posttranslational modification (PTM). These DNA modifications in fixed cells are labeled with respective DNA barcoding probes, and then the PTM site is tethered with a DNA walking probe. Cell-TALKING can continuously produce cleavage records of any barcoding probes nearby the walking probe. New 3'-OH ends are generated on the cleaved barcoding probes to induce DNA amplification for downstream detections. Combining fluorescence imaging, we identify various combinatorial chromatin modifications and investigate their dynamic changes during cell cycles. We also explore the nanoenvironments in different cancer cell lines and clinical specimens. In principle, using high-throughput sequencing instead of fluorescence imaging may allow the detection of complex cellular nanoenvironments containing tens of biomolecules such as transcription factors.

Pairwise Proximity-Differentiated Visualization of Single-Cell DNA Epigenetic Marks.

Spatial positioning and proximity of relevant biomolecules such as DNA epigenetic marks are fundamental to a deeper understanding of life. However, it remains poorly explored and technically challenging. Here we report the pairwise proximity-differentiated visualization of single-cell 5-formylcytosine (5fC) and 5-hydroxymethylcytosine (5hmC). These two marks on chromatin in fixed cells are successively labeled and crosslinked with their DNA primer probes via click chemistry. Based on a pairwise proximity-differentiated mechanism, proximal 5fC/5hmC sites and residual 5fC or 5hmC sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. We thus demonstrate the differentiated visualization of 5fC or 5hmC spatial positioning and their pairwise proximity in single cells. Such multi-level subcellular information may provide insights into regulation functions and mechanisms of chromatin modifications, and the spatial proximity can expose the potential crosstalk or interaction between their reader proteins.

Aptamer-Functionalized Microdevices for Bioanalysis.

Aptamers have drawn great attention in the field of biological research and disease diagnosis for the remarkable advantages as recognition elements. They show unique superiority for facile selection, desirable thermal stability, flexible engineering, and low immunogenicity, complementing the use of conventional antibodies. Aptamer-functionalized microdevices offer promising properties for bioanalysis applications because of the compact sizes, minimal reaction volume, high throughput, operational feasibility, and controlled preciseness. In this review, we first introduce the innovative technologies in the selection of aptamers with microdevices and then highlight some advanced applications of aptamer-functionalized microdevices in bioanalysis field for diverse targets. Aptamer-functionalized microfluidic devices, microarrays, and paper-based and other interface-based microdevices are all bioanalysis platforms with huge potential in the near future. Finally, the major challenges of these microdevices applied in bioanalysis are discussed and future perspectives are also envisioned.

Nucleic Acids Analysis.

Nucleic acids are natural biopolymers of nucleotides that store, encode, transmit and express genetic information, which play central roles in diverse cellular events and diseases in living things. The analysis of nucleic acids and nucleic acids-based analysis have been widely applied in biological studies, clinical diagnosis, environmental analysis, food safety and forensic analysis. During the past decades, the field of nucleic acids analysis has been rapidly advancing with many technological breakthroughs. In this review, we focus on the methods developed for analyzing nucleic acids, nucleic acids-based analysis, device for nucleic acids analysis, and applications of nucleic acids analysis. The representative strategies for the development of new nucleic acids analysis in this field are summarized, and key advantages and possible limitations are discussed. Finally, a brief perspective on existing challenges and further research development is provided.