The development of novel synthesis methods for magnetic beads is paramount to advancing nucleic acid extraction in the field of in vitro diagnostics. In this study, monodisperse magnetic polymer beads were synthesized via a pH-induced in-situ co-precipitation method using poly (styrene-acrylic acid) microspheres as templates. The poly(styrene-acrylic acid) microspheres were loaded with a high density of superparamagnetic iron oxide nanoparticles due to their carboxyl-rich surfaces. The morphology and properties of the obtained magnetic polymer beads were evaluated by scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry, X-ray diffraction testing, and so on. The average size of the as-prepared magnetic polymer beads was 670 nm in diameter with a narrow size distribution. The saturation magnetization and magnetic content of the resultant magnetic polymer beads were 23.52 emu/g and 29%, respectively. After being coated with a silicon oxidation layer, the magnetic polymer beads demonstrated successful DNA adsorption via silanol-DNA interactions under chaotropic conditions, with a recovery efficiency ranging from 85.4% to 95.8% for model DNA and a capture capacity of 12.8 mg/g. Due to the stable adsorption of DNA onto the bead surface, the resulting DNA-bead complexes can be efficiently separated from the solution using an external magnetic field, achieving complete collection within 20 s. The beads were further employed for purification of cell-free DNA (cfDNA) from plasma, yielding an average of 22.56 ng from 2 mL of plasma.

Self-assembled DNA crystals provide highly ordered three-dimensional frameworks with nanoscale precision for advanced functional materials. However, conventional concentration gradient-driven droplet crystallization in open environments suffers from poorly controlled supersaturation, laborious operation, and heterogeneous products. Here, we report a homogeneous solution strategy for DNA crystal assembly in a closed test tube through base sequence regulation and chemical modification. Rational tuning of the base composition of DNA building blocks and sticky-end 5 '-phosphorylation modification enables rapid crystallization within 2 h with relatively uniform crystal sizes. Additionally, phosphorothioate backbone modification enables the assembly of large-sized DNA crystals in a homogeneous environment. Due to the fully enclosed and compositionally uniform reaction environment, this strategy affords highly reproducible control over the crystal size and morphology across batches. This homogeneous solution-based crystallization platform provides a general route to DNA crystalline materials, laying a materials foundation for the construction of functional devices.

Extracellular vesicles (EVs) play an important role in many physiological processes, and surface labeling is essential for the research and application of EVs. Fluorescence probes provide a sensitive and visual tool for EV labeling and imaging; however, most of them suffer from some native shortcomings, e.g., false positives caused by the aggregation of hydrophobic organic dyes and membrane destruction in inorganic phosphor labeling. To resolve these issues, in this work, we presented an emerging fluorescent probe, i.e., cholesteryl-modified ultrabright green emissive carbon dots (Chol-GCDs), for multivalent labeling and further highly sensitive detection of EVs. GCDs revealed a high quantum yield (65%), abundant amino groups, and good water solubility. Through a covalent linking, cholesteryl was efficiently modified on the surface of CDs, achieving the construction of a fluorescence probe, i.e., Chol-GCDs, for EV recognition. Chol-GCDs have shown great potential for multivalent and stable EV labeling due to their advantages of plentiful grafting of cholesteryl, ultrasmall size, water solubility, and good tolerance to photobleaching. Thereafter, a sandwich-model fluorescence detection platform (96-well plates) for EVs was constructed. This platform enabled quantitative EV detection over a wide range from 103 to 106 particles/mL and demonstrated good analytical performance in biological samples such as serum and cell culture supernatants. This work provided a promising and competitive fluorescence label for EV labeling and would open an opportunity to exploit a versatile platform for the research and applications relevant with EVs.

Cells execute complex biological functions through programmed biomolecular condensation, yet a general and programmable strategy for nucleic acid condensation in vitro remains challenging. Here, we demonstrate that carbon dots (CDs) function as an electrostatic-driven versatile chemical compiler that translates nucleobase sequence into programmed condensate architectures and functions. We found that citric acid-ethylene diamine carbon dots (CA-EDA CDs) drive the condensation of both ssDNA and mRNA via multiple weak interactions. Crucially, the spatially heterogeneous electrostatic potential on the CD surface decodes the distinct electrostatic properties of nucleobases through Coulomb-dominated interactions, establishing a definitive binding hierarchy (G > C > T > A), as revealed by systematic binding studies and molecular dynamics simulations. Based on this chemical compiler, we can achieve hierarchical control of the condensate structure. Furthermore, by programming condensate density via sequence engineering, we achieved switchable up- and down-regulation of GFP mRNA translation in a cell-free system, faithfully mimicking the regulatory role of natural ribonucleoprotein granules. This work establishes CDs as a versatile compiler platform for programmable nucleic acid condensation, transcending the limitations of nonspecific electrostatic neutralization and paving the way for constructing intelligent biomaterials and synthetic biological systems with encoded function.

Nonenzymatic isothermal nucleic acid self-assembly techniques (e.g., the hybridization chain reaction, HCR) hold potential in building materials and biological sensing. However, a traditional HCR is triggered by the random diffusion and disordered conformations of ssDNA initiators, resulting in asynchronous initiation and inherently highly heterogeneous products that do not meet the standards of well-defined nanomaterials. Herein, we developed a nanomechanical restricted strategy directed by tetrahedral DNA frameworks (TDFs) to control HCR self-assembly. We found that the restricted initiator at TDF vertices could induce DNA hairpin assembly to form homogeneous products in solution. Mechanistically, we found that TDFs accelerated the strand displacement rate of the starting H1 and synchronized the assembly process of the HCR. Furthermore, the TDF exhibited strict vertex specificity for HCR controllable assembly, and side extension of the initiator could not result in homogeneous products. This work presents a straightforward and efficient approach for controlling the living self-assembly of macromolecular DNA, thus providing a novel tool for HCR-based nanomanufacturing and quantitative sensing applications.

Luminescence-based analytical methods have attracted considerable attention due to the fascinating merits of high sensitivity, affordability, and quick screening. However, autofluorescence and scattered light caused by the excitation of ultraviolet (UV) or blue light result in low ratio of signal to noise (S/N) or sensitivity. The adoption of diverse phosphors with unique characters e.g. near-infrared (NIR) excitation or emission, time-resolution has partially resolved the above shortcomings, but matrix effects are still critical. The introduction of magnetic separation is an ideal and promising solution. Magnetic-luminescence bifunctional nano-beads have achieved great advancement both in probe design and construction, and versatile assay applications, but are rarely summarized and discussed. Herein, in this review, we systematically summarize recent research progress on the synthesis methodologies of magnetic-luminescence bifunctional probes, and their applications in detection and testing. We first classify their fabrications strategies, meanwhile, and the corresponding merits and drawbacks are discussed objectively. Their various applications in lateral flow immunoassay (LFIA), fluorescent encoding, molecular imprinting, metal ion detection, and biomedical imaging are summarized and discussed. Finally, the challenges and future possibilities of magnetic-luminescence nano-beads are proposed, and we hope that it will bring some inspirations to the relevant researchers.

Triple-negative breast cancer (TNBC) is highly invasive with a poor prognosis, and chemotherapy remains the clinical treatment of choice. Paclitaxel is a commonly used first-line chemotherapy drug, but its untargeted distribution poses clinical challenges. Inspired by antibody-drug conjugates, we develop a precisely structured framework nucleic acid-programmed aptamer-paclitaxel conjugate (FAPC) with chemically well-defined paclitaxel loading dosing, enabling the regulation of receptor-aptamer affinity to facilitate tumor-targeted chemotherapy. Utilizing framework nucleic acids as a precise addressing scaffold, we organize the AS1411 aptamer with accurate intermolecular spacing and find that an inter-aptamer spacing of 19.04 nm could enhance the affinity of the FAPC for tumor cells. Then, the multifunctional FAPC can disrupt actin reorganization to achieve cytotoxicity in tumor cells. Furthermore, the AS1411-specifically modified FAPC further enhances the structure-dependent selective accumulation of drugs at tumor sites in a human xenograft model of triple-negative breast cancer, subsequently leading to significantly improved antitumor efficacy and reduced toxicity. The FAPC provides a precisely programmable platform for efficient targeted delivery of chemotherapeutic agents to malignancies.

The precise spatial organization of cells into functional tissues represents a fundamental challenge in biology and regenerative medicine. Conventional methods for directing cell assembly often lack the specificity, reproducibility, and dynamic control necessary to mimic native tissue architectures. This review explores the emerging use of DNA as a programmable and biocompatible strategy to engineer cell–cell interactions and construct hierarchically ordered tissue models. We first introduce the properties of various DNA toolbox and their strategies for cell modification and assembly. Importantly, we highlight the latest research advances in DNA-encoded cell spheroids, layered tissues, and organoids. Finally, we summarize current challenges and future directions in DNA-programmed assembly.

Bacterial biofilms and their microenvironment are significant challenges that must be faced in the design of antibacterial drugs. Microenvironment-responsive mimetic peroxidases (POD) have been demonstrated to be an efficient solution to eliminating bacterial biofilms. However, they inevitably require additional H2O2 and/or acid due to the poor permeabilities towards biofilms. Herein, we report POD-like copper-doped carbon dots (named CuCD1) synthesized through a facile microwave-assisted carbonization manner. The characteristics of ultrasmall size (< 5 nm) and positive charge enabled it to possess good penetrability toward bacterial biofilm. As expected, CuCD1 showed great damage to bacteria due to the generation of hydroxyl radicals (center dot OH), which originated from the catalytic decomposition of endogenous H2O2 under a weak acid bacterial biofilm microenvironment. This highly increased oxidative stress resulted in the alteration of cell membrane permeability, subsequent cell death, and the final eradication of bacterial biofilm and the exposed bacteria. Moreover, to verify the practicality in vivo, CuCD1 was introduced to a routine hydrogel that was crosslinked by carboxymethyl chitosan (CMCS) and oxidized dextran (ODEX). In comparison with the control groups, the composite hydrogel, i.e., CuCD1-CMCS-ODEX revealed better antibacterial performance and thus accelerated wound healing and collagen disposition. This work would open opportunities to design CDs-based biofilm microenvironment-responsive antibacterial nanoagents.

Exploring novel synthesis strategies for magnetic beads to extract nucleic acids is of great significance in the field of in vitro diagnostics. In the present research, monodisperse magnetic mesoporous silica beads were synthesized via the thermolysis reaction of Fe(acac)3 by using large-pore dendritic silica colloids as templates, and were further functionalized with a highly pH-sensitive histidine-glutamate co-oligopeptide for deoxyribonucleic acid extraction. The large-pore dendritic silica colloid scaffolds were utilized for high-density incorporation of superparamagnetic iron oxide nanoparticles within the vertical channels. The morphology and properties of the as-prepared pH-sensitive oligopeptide magnetic mesoporous silica beads were evaluated by transmission electron microscopy, scanning electron microscopy, vibrating sample magnetometry, X-ray photoelectron spectroscopy, X-ray diffraction testing and so on. The average size of the obtained magnetic beads was 370 nm in diameter with a narrow size distribution. The saturation magnetization and magnetic content of the resultant magnetic beads were 25 emu g-1 and 59%, respectively. Moreover, the magnetic mesoporous silica beads exhibited an obvious pH-responsive behavior. Due to these remarkable features, successful deoxyribonucleic acid capture using the as-prepared pH-sensitive oligopeptide magnetic mesoporous silica beads was achieved.

Idiopathic pulmonary fibrosis (IPF) is an irreversible and fatal lung disease characterized by persistent alveolar epithelial cell injury and extracellular matrix deposition. Early dual modulation of oxidative stress and inflammation may offer a promising therapeutic opportunity. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) offer therapeutic promise but face challenges in scalability and efficient lung delivery. Here, we developed a biomimetic extracellular vesicle-spherical nucleic acid (BEV-SNA) platform for IPF therapy. BEV-SNA were constructed by integrating mechanically extruded BEVs from primary MSCs with cholesterol-modified ssDNA through hydrophobic co-assembly. In stemness-maintained P0-P1 MSCs, the production of BEVs increased by 17.2-fold compared to natural EVs. Benefiting from a three-dimensionally dense and negatively charged DNA shell, BEV-SNA reduce airway adhesion, enabling deep pulmonary delivery and efficient cellular uptake. In IPF models, BEV-SNA demonstrated multiphase therapeutic effects, including protection of alveolar epithelial cells from ROS, anti-inflammatory activity, and late-stage anti-fibrotic action, effectively halting fibrosis progression and achieving a 50% survival rate in mice. This study presents a novel therapeutic platform combining the natural biomimicry of EVs with the functional adaptability of SNAs, proposing an innovative strategy for pulmonary drug delivery and the treatment of respiratory diseases.

Extracellular vesicles (EVs), as natural mediators of intercellular communication, hold substantial promise for diagnostics, drug delivery, and regenerative medicine. However, their translation remains constrained by vulnerability to hostile microenvironments, rapid clearance with a short in vivo half‐life, and limited control over localization and dosing. Encapsulation based on multi‐scale materials engineering can endow EVs with programmable release, environmental responsiveness, and site‐specific delivery. In this review, recent advances in EV encapsulation technologies are synthesized. Guided by structural design principles, encapsulation is classified into three scales comprising nanoscale, microscale, and macroscale, and each scale provides distinct mechanisms for protection and controlled release. A comparative overview of representative strategies is then offered, and their advantages and application contexts are summarized. Finally, key challenges and future directions are outlined, including elucidation of material–EV interactions, development of scalable and standardized manufacturing, and realization of on‐demand, spatiotemporally precise release in response to physiological cues.

Female germline stem cells (FGSCs) that can postnatally produce new oocytes hold promise for reproductive and regenerative medicine. However, the maintenance of their in vitro stemness and the active control of the quiescence-to-activation transition pose limits on their applications. Here, we develop DNA hydrogel-based artificial 3D extracellular matrices to direct the fate of mouse- and monkey-derived FGSCs. We demonstrate a hierarchical DNA crosslinking strategy to program the stiffness of DNA hydrogels, independent of their chemical composition and stress relaxation rate. The improved hydrogel stiffness promotes cell spreading and proliferation. Remarkably, FGSCs encapsulated in the low-stiffness DNA hydrogel (storage modulus 270 Pa) maintain a quiescent state, whereas the removal of hydrogels reactivates FGSCs. This fate transition is dependent on mechano-induced actin polymerization and Yes-associated protein signaling. Finally, we demonstrate that mouse FGSCs released from the DNA hydrogel can develop into oocytes, embryos, and ultimately healthy offspring.

Fluorescence lateral flow immunoassays (FL-LFIA) have attracted considerable attention in clinical diagnosis due to their outstanding merits of affordable, sensitive, on-site, and quick detection. However, they are still plagued by significant signal interference, such as autofluorescence and scattered light. The development of high-performance and robust phosphors, i.e., label probes featuring with the character of low/no optical background, remains a great challenge. Herein, we report a novel visualized phosphorescence LFIA (Phos-LFIA), where the composite microspheres, i.e., carbon dots (CDs) covalently embedded in dendritic mesoporous silicon nanoparticles (DMSNs), were designed and selected as the report probes. The obtained CDs@DMSNs revealed uniform morphologies and particle sizes, as well as ultralong (lifetime: 1.14 s, visible for over 8 s to naked eyes) room temperature phosphorescence (RTP) in aqueous solution. As competitive nanotags, CDs@DMSNs were designed for an ultralong phosphorescence-based time-gated LFIA for cardiac troponin I (cTnI) without optical interference. The fabricated Phos-LFIA test strips demonstrated zero-background signal and were applied for highly sensitive cTnI detection in both buffer and a complex serum matrix, with corresponding limits of detection (LODs) of 0.19 and 0.21 ng/mL, respectively. For a clinical validation, the proposed Phos-LFIA revealed an excellent clinical analytical performance (sensitivity: 95.45%, specificity: 88.9%, κ value: 0.85), demonstrating its potential for rapid and accurate diagnosis of myocardial infarction. This work provided a promising background-free probe for FL-LFIA, and it would also open an opportunity for developing highly sensitive screening platforms for other targets through modifying different recognition ligands onto CDs@DMSNs.

The lateral flow immunoassay (LFIA), as a popular point-of-care-testing (POCT) technology, has attracted increasing attention, but its analytical performance, especially detection sensitivity, relies on the photophysical properties of the nanotags used. The exploitation of high-performance, robust tags featuring facile fabrication and excellent water solubility remains a great challenge. Herein, we reported a one-pot hydrothermal preparation of diverse polymers (e.g., PAA, PEI, and PVA)-modified YVO4:Eu nanoparticles (NPs). The polymer-coated YVO4:Eu NPs revealed abundant surface functional groups (e.g., carboxyl, amino, and hydroxyl), good water solubility, excellent colloidal stability, and strong red emission (absolute quantum yield over 50%). To demonstrate their potential in biolabeling, a routine sandwich LFIA was designed for quick detection of procalcitonin (PCT) using PAA-YVO4:Eu as the nanotags. Through the luminescence image collection and the corresponding RGB color value transformation, the LFIA test strips were applied for a smartphone-based portable and quantitative detection of PCT (0.2-50 ng/mL) with a limit of detection of 0.06 ng/mL in buffer. Especially, the same quantitative fitting was obtained in human serum with a slightly higher detection limit (0.16 ng/mL), demonstrating high robustness of the test strips in a complex biological matrix. For a clinical validation, in comparison with the chemiluminescent immunoassay, the proposed test strips revealed a good analytical performance (sensitivity: 88.9%, specificity: 95.2%, kappa value: 0.83). This work also provides an opportunity to build versatile LFIA platforms for other targets using the developed YVO4:Eu probes.

The functionalization of living cells, both internally and externally, transforming them into micromachines with specified functions, holds significant potential in fields such as biosensing, biocomputing, and intelligent theranostics. However, due to the complexity and dynamic nature of living cells, it remains challenging to allocate exogenous functional materials to specific locations within the cell or on its surface and maintain their positions stable for a reasonable period. Here, we devise a DNA -programmed cargo distributing system (DCD), capable of distributing functional modules to the cell membrane or within the cell as needed. This system includes an amphiphilic DNA structure for determining the destination of the cargo and a DNA connector carried on it for recognizing the DNA -encoded cargo. We test three different morphologies of amphiphilic DNA structures and find that their efficiencies in cell surface retention and cell internalization significantly varied, enabling the distribution of nanoparticle cargos on the cell membrane and within the cell in distinct proportions. Their positions can remain stable for at least 6 h. Moreover, this allocation method shows specificity, which minimizes the deployment of mismatched cargo. This method provides new tools for the modular construction of cellular micromachines.

BACKGROUND:Methotrexate (MTX) serves as the initial treatment for rheumatoid arthritis (RA). However, a substantial proportion of RA patients, estimated between 30% and 50%, do not respond positively to MTX. While the T-cell receptor (TCR) is crucial for the immune response during RA, its role in differentiating MTX responsiveness has not been thoroughly investigated. METHODS:This study used next-generation sequencing to analyze the TCR β-chain complementary determining region sequences in peripheral blood mononuclear cells obtained from RA patients before MTX treatment. This study aimed to compare the characteristics of the TCR repertoire between the MTX responder and non-responder groups. RESULTS:The study identified a significant difference in the TRBV6-6 gene (p = .003) concerning MTX treatment response. Additionally, a significant difference was found in the number of "3" nucleotide deletions at the 5'Jdels site (p = .023) in the VDJ rearrangement. CONCLUSION:These findings suggest distinct TCR repertoire characteristics between MTX responder and non-responder groups among RA patients. This discovery offers new insights into understanding the variable responses of RA patients to MTX therapy.