Accelerated diagnosis: a crosslinking catalytic hairpin assembly system for rapid and sensitive SARS-CoV-2 RNA detection.

The COVID-19 pandemic has underscored the urgent need for rapid and reliable strategies for early detection of SARS-CoV-2. In this study, we propose a DNA nanosphere-based crosslinking catalytic hairpin assembly (CCHA) system for the rapid and sensitive SARS-CoV-2 RNA detection. The CCHA system employs two DNA nanospheres functionalized with catalytic hairpin assembly (CHA) hairpins. The presence of target SARS-CoV-2 RNA initiated the crosslinking of DNA nanospheres via CHA process, leading to the amplification of fluorescence signals. As a result, the speed of SARS-CoV-2 diagnosis was enhanced by significantly increasing the local concentration of the reagents in a crosslinked DNA product, leading to a detection limit of 363 fM within 5 min. The robustness of this system has been validated in complex environments, such as fetal bovine serum and saliva. Hence, the proposed CCHA system offers an efficient and simple approach for rapid detection of SARS-CoV-2 RNA, holding substantial promise for enhancing COVID-19 diagnosis.

Three-Dimensional CHA-HCR System Using DNA Nanospheres for Sensitive and Rapid Imaging of miRNA in Live Cells and Tissues.

Isothermal, enzyme-free amplification techniques, such as the hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), have gained increasing attention for miRNA analysis. However, current methodological challenges, including slow kinetics, low amplification efficiency, difficulties in efficient cellular internalization of DNA probes, and concerns regarding the intracellular stability of nucleic acids, need to be addressed. To this end, we propose a novel strategy for sensitive miRNA detection based on a three-dimensional (3D) CHA-HCR system. This system comprises two DNA nanospheres, named DS-13 and DS-24, which are functionalized with CHA and HCR hairpins. Target miR-21 initiates CHA between the two nanospheres, thereby activating downstream HCR and bringing cyanine 3 (Cy3) and cyanine 5 (Cy5) into proximity. The 3D CHA-HCR process leads to the formation of large DNA aggregates and the generation of fluorescence resonance energy transfer signals. In this strategy, the employment of a cascaded reaction and spatial confinement effect improve sensitivity and kinetics, while the use of DNA nanocarriers facilitates cellular delivery and protects nucleic acid probes. The experimental results in vitro, in living cells, and in clinical tissue samples demonstrated the desirable sensing performance. Collectively, this approach holds promise as a valuable tool for cancer diagnosis and biomedical research.

Hierarchical MOF@AuNP/Hairpin Nanotheranostic for Enhanced Photodynamic Therapy via O2 Self-Supply and Cancer-Related MicroRNA Imaging In Vivo.

Developing a nanotheranostic with a high sensing performance and efficient therapy was significant in cancer diagnosis and treatment. Herein, a Au nanoparticle and hairpin-loaded photosensitive metal-organic framework (PMOF@AuNP/hairpin) nanotheranostic was constructed by growing AuNPs on PMOF in situ and then attaching hairpins. On the one hand, the PMOF@AuNP/hairpin nanotheranostic could effectively transfer O2 into ROS, facilitating efficient PDT. Additionally, the nanotheranostic possessed catalase-like activity, which could effectively catalyze H2O2 to generate O2, thus achieving O2-evolving PDT and significantly enhancing the antitumor effect of PDT in vivo. On the other hand, the nanotheranostic showed a high loading efficiency of hairpins and achieved the sensitive and selective detection of miR-21 both in living cells and in vivo. Moreover, the nanotheranostic could dynamically monitor the miR-21 level. Due to the excellent imaging performance, the nanotheranostic could recognize cancer cells and might provide important information on cancer progression for PDT. The developed PMOF@AuNP/hairpin nanotheranostic provided a useful tool for tumor diagnosis and antitumor therapy.

Self-assembly of molecular beacons through metal ion coordination for fluorescence imaging of miRNA in living cells.

Fluorescence nanosensors based on functional nucleic acids have been explored as a powerful sensing platform for disease-relevant miRNAs. This work developed a new hybrid nanosensor (Zr-B) through coordination-driven self-assembly of Zr ions and beacons. The prepared nanosensor exhibited high loading efficiency of beacons and could achieve sensitive and specific detection for miRNAs. The hybrid nanosensor could transfer beacons into living cells efficiently and maintain high stability and biocompatibility in the biological environment, achieving effective miRNA fluorescence imaging in living cells. Therefore, the resultant nanosensor holds potential for applications in disease diagnostics.

A novel 1,8-naphthalimide-based fluorescent chemosensor for the detection of HSA in living cells.

Human serum albumin (HSA) is an essential protein for maintaining human health. Accurate detection and quantification of HSA are of great significance for disease diagnosis and biochemical research. Here, a new HSA fluorescent probe BNPE based on the 1,8-naphthalimide fluorophore was designed and synthesized. The probe could recognize HSA through a twisted intramolecular charge transfer mechanism, effectively avoid the interference of most substances, and realize HSA fluorescence imaging in living cells.

Aptamer-Based Logic Computing Reaction on Living Cells to Enable Non-Antibody Immune Checkpoint Blockade Therapy.

Precise and lasting immune checkpoint blockade (ICB) therapy with high objective response rate remains a significant challenge in clinical trials. We thus report the development of an aptamer-based logic computing reaction to covalently conjugate immune checkpoint antagonizing aptamers (e.g., aPDL1 aptamer) on the surface of cancer cells, achieving effective and sustained ICB therapy without the need for antibodies. Specifically, azides were metabolically labeled on the cell-surface glycoproteins as "chemical receptors", enabling cyclooctyne-coupling aPDL1 aptamers to achieve aptamer-based logic computing-mediated azides/cyclooctynes-based bioorthogonal reaction. In stepwise fashion, PDL1 plus azide-bearing glycoproteins are expressed on cells and become multiple inputs in accordance with Boolean logic. Then, if the "AND" conditions of the algorithm are met, cyclooctyne-coupling aptamers are conjugated on the living cell surface, significantly prolonging overall mouse survival by triggering a precise and sustained T cell-mediated antitumor immunotherapy, otherwise not. Our findings indicate that DNA logic computing-mediated cyclooctyne/azide-based bioorthogonal reaction can improve the precision and robustness of ICB therapy, thereby potentially improving the objective response rate.

Imaging Intracellular S-Adenosyl Methionine Dynamics in Live Mammalian Cells with a Genetically Encoded Red Fluorescent RNA-Based Sensor.

To understand the role of intracellular metabolites in cellular processes, it is important to measure the dynamics and fluxes of small molecules in living cells. Although conventional metabolite sensors composed of fluorescent proteins have been made to detect some metabolites, an emerging approach is to use genetically encoded sensors composed of RNA. Because of the ability to rapidly generate metabolite-binding RNA aptamers, RNA-based sensors have the potential to be designed more readily than protein-based sensors. Numerous strategies have been developed to convert the green-fluorescent Spinach or Broccoli fluorogenic RNA aptamers into metabolite-regulated sensors. Nevertheless, red fluorescence is particularly desirable because of the low level of red background fluorescence in cells. However, the red fluorescent variant of the Broccoli aptamer, Red Broccoli, does not exhibit red fluorescence in cells when imaged with its cognate fluorophore. It is not known why Red Broccoli is fluorescent in vitro but not in live mammalian cells. Here, we develop a new fluorophore, OBI (3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime-1-benzoimidazole), which binds Red Broccoli with high affinity and makes Red Broccoli resistant to thermal unfolding. We show that OBI enables Red Broccoli to be readily detected in live mammalian cells. Furthermore, we show that Red Broccoli can be fused to a S-adenosyl methionine (SAM)-binding aptamer to generate a red fluorescent RNA-based sensor that enables imaging of SAM in live mammalian cells. These results reveal a red fluorescent fluorogenic aptamer that functions in mammalian cells and that can be readily developed into red fluorescent RNA-based sensors.

In Vivo Monocyte/Macrophage-Hitchhiked Intratumoral Accumulation of Nanomedicines for Enhanced Tumor Therapy.

The inner region of solid tumors is found to be high-pressure, hypoxic, and immunosuppressive, providing a breeding ground for tumor aggressiveness and metastasis. While intratumoral accumulation of nanomedicines combined with immunomodulation would significantly enhance therapeutic efficacy, such potential is challenged by the compressed environment and distinct heterogeneity of the tumor bulk. By using an apoptotic body (AB) as the carrier, we develop an effective and universal intratumoral nanomedicine delivery system for the long-lasting remission of tumors. Our results show that the AB-encapsulated nanomedicine (using CpG immunoadjuvant-modified gold-silver nanorods as a model), after intravenous injection, can be specifically phagocytosed by inflammatory Ly-6C+ monocytes, which then actively infiltrate the tumor center via their natural tumor-homing tendency. With the integration of AB-facilitated intratumoral accumulation, the nanorod-based photothermal effect, and CpG-promoted immunostimulation, this cell-mediated delivery system can not only efficiently ablate primary tumors but also elicit a potent immunity to prevent tumors from metastasizing and recurring.

Hypoxia-Activated PEGylated Conditional Aptamer/Antibody for Cancer Imaging with Improved Specificity.

Aptamers and antibodies, as molecular recognition probes, play critical roles in cancer diagnosis and therapy. However, their recognition ability is based on target overexpression in disease cells, not target exclusivity, which can cause on-target off-tumor effects. To address the limitation, we herein report a novel strategy to develop a conditional aptamer conjugate which recognizes its cell surface target, but only after selective activation, as determined by characteristics of the disease microenvironment, which, in our model, involve tumor hypoxia. This conditional aptamer is the result of conjugating the aptamer with PEG5000-azobenzene-NHS, which is responsive to hypoxia, here acting as a caging moiety of conditional recognition. More specifically, the caging moiety is unresponsive in the intact conjugate and prevents target recognition. However, in the presence of sodium dithionite or hypoxia (<0.1% O2) or in the tumor microenvironment, the caging moiety responds by allowing conditional recognition of the cell-surface target, thereby reducing the chance of on-target off-tumor effects. It is also confirmed that the strategy can be used for developing a conditional antibody. Therefore, this study demonstrates an efficient strategy by which to develop aptamer/antibody-based diagnostic probes and therapeutic drugs for cancers with a unique hypoxic microenvironment.

Smart Nanodrug with Nuclear Localization Sequences in the Presence of MMP-2 To Overcome Biobarriers and Drug Resistance.

A series of physiological barriers have impeded nanoparticle-based drug formulations (NDFs) from reaching their targeted sites and achieving therapeutic outcomes. In this study, we develop size-controllable stealth doxorubicin-loaded nanodrug coated with CD47 peptides (DOX/sNDF-CD47) based on supramolecular chemistry to overcome multiple biological barriers. The smart DOX/sNDF-CD47 can efficiently decrease sequestration by macrophages and disassemble into poly(amidoamine) dendrimers with nuclear localization sequences (DOX/PAMAM-NLS) in the presence of matrix metalloproteinase-2 (MMP-2). Such structure transformation endows DOX/sNDF-CD47 with the ability of deep penetration in multicellular tumor spheroid, lysosomal escape, and nucleus localization, resulting in excellent cytotoxicity and drug resistance combating. In vivo experiments further confirmed that DOX/sNDF-CD47 has good tumor-targeting ability and can significantly improve therapeutic efficacy of DOX on xenograft tumor model. The ability to overcome multiple biological barriers makes sNDF-CD47 a promising NDFs to treat cancer expressing MMP-2 and combating drug resistance.

Circular Bivalent Aptamers Enable in Vivo Stability and Recognition.

Aptamers are powerful candidates for molecular imaging and targeted therapy of cancer based on such appealing features as high binding affinity, high specificity, site-specific modification and rapid tumor penetration. However, aptamers are susceptible to plasma exonucleases in vivo. This seriously affects their in vivo applications. To overcome this key limitation, we herein report the design and development of circular bivalent aptamers. Systematic studies reveal that cyclization of aptamers can improve thermal stability, nuclease resistance and binding affinity. In vivo fluorescence imaging further validates the efficient accumulation and retention of circular bivalent aptamers in tumors compared to "mono-aptamers". Therefore, this study provides a simple and efficient strategy to boost in vivo aptamer applications in cancer diagnosis and therapy.

Aptamer-integrated DNA nanostructures for biosensing, bioimaging and cancer therapy.

The combination of nanostructures with biomolecules leading to the generation of functional nanosystems holds great promise for biotechnological and biomedical applications. As a naturally occurring biomacromolecule, DNA exhibits excellent biocompatibility and programmability. Also, scalable synthesis can be readily realized through automated instruments. Such unique properties, together with Watson-Crick base-pairing interactions, make DNA a particularly promising candidate to be used as a building block material for a wide variety of nanostructures. In the past few decades, various DNA nanostructures have been developed, including one-, two- and three-dimensional nanomaterials. Aptamers are single-stranded DNA or RNA molecules selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), with specific recognition abilities to their targets. Therefore, integrating aptamers into DNA nanostructures results in powerful tools for biosensing and bioimaging applications. Furthermore, owing to their high loading capability, aptamer-modified DNA nanostructures have also been altered to play the role of drug nanocarriers for in vivo applications and targeted cancer therapy. In this review, we summarize recent progress in the design of aptamers and related DNA molecule-integrated DNA nanostructures as well as their applications in biosensing, bioimaging and cancer therapy. To begin with, we first introduce the SELEX technology. Subsequently, the methodologies for the preparation of aptamer-integrated DNA nanostructures are presented. Then, we highlight their applications in biosensing and bioimaging for various targets, as well as targeted cancer therapy applications. Finally, we discuss several challenges and further opportunities in this emerging field.

Functional nucleic acid-based hydrogels for bioanalytical and biomedical applications.

Hydrogels are crosslinked hydrophilic polymers that can absorb a large amount of water. By their hydrophilic, biocompatible and highly tunable nature, hydrogels can be tailored for applications in bioanalysis and biomedicine. Of particular interest are DNA-based hydrogels owing to the unique features of nucleic acids. Since the discovery of the DNA double helical structure, interest in DNA has expanded beyond its genetic role to applications in nanotechnology and materials science. In particular, DNA-based hydrogels present such remarkable features as stability, flexibility, precise programmability, stimuli-responsive DNA conformations, facile synthesis and modification. Moreover, functional nucleic acids (FNAs) have allowed the construction of hydrogels based on aptamers, DNAzymes, i-motif nanostructures, siRNAs and CpG oligodeoxynucleotides to provide additional molecular recognition, catalytic activities and therapeutic potential, making them key players in biological analysis and biomedical applications. To date, a variety of applications have been demonstrated with FNA-based hydrogels, including biosensing, environmental analysis, controlled drug release, cell adhesion and targeted cancer therapy. In this review, we focus on advances in the development of FNA-based hydrogels, which have fully incorporated both the unique features of FNAs and DNA-based hydrogels. We first introduce different strategies for constructing DNA-based hydrogels. Subsequently, various types of FNAs and the most recent developments of FNA-based hydrogels for bioanalytical and biomedical applications are described with some selected examples. Finally, the review provides an insight into the remaining challenges and future perspectives of FNA-based hydrogels.

Enhancing RNA detection and breast cancer subtyping with a universal 3D-hybridization chain reaction system.

Breast cancer, a globally prevalent malignancy, is characterized by pronounced heterogeneity. Accurate subtyping requires the simultaneous detection of different biomarkers, which is crucial for personalized treatment strategies. However, existing methodologies are hindered by limited versatility and sensing performance. To overcome these hurdles, this study presents a universal 3D-Hybridization Chain Reaction (3D-HCR) system for RNA detection and subtype-specific diagnosis of breast cancer. The system integrated a universal trigger for HCR, thereby circumventing the need for complex sequence design and enabling the analysis of various RNA targets. Leveraging the spatial-confinement effect offered by DNA nanocarriers, this system exhibited superior amplification efficiency, achieving detection limits of 3.83 pM and 4.96 pM for PD-L1 mRNA and miR-21, respectively. Importantly, the system could differentiate between triple-negative breast cancer and estrogen receptor-positive breast cancer in both living cells and clinical tissues. These findings underscore the potential of the universal 3D-HCR system as a promising tool in clinical diagnostics. With its proven proficiency in breast cancer diagnostics and versatility in RNA analysis, this system holds the promise of broadening the horizons of precision medicine.

Fe/Cu-AuNP nanocomposites as enzyme-like catalysts to modulate the tumor microenvironment for enhanced synergistic cancer therapy.

The intensive investigation of chemodynamic therapy (CDT) for tumor eradication revealed that the therapeutic effects of this ROS-mediated therapy are limited by endogenous reductants and inefficient Fenton-like reactions. In this study, we developed a new Fe/Cu-AuNP-PEG nanocomposite to enhance CDT and provide a synergistic treatment for tumors. The Fe/Cu-AuNP-PEG nanocomposite demonstrated effective ˙OH production and high photothermal conversion efficiency under 808 nm illumination, which promoted the ˙OH production, thereby enhancing the CDT efficacy and exhibiting a synergistic treatment for cancer. More importantly, the Fe/Cu-AuNP-PEG nanocomposite showed the ability to deplete GSH and catalyze glucose to generate H2O2, which facilitated the Fenton-like reaction and reduced the antioxidant properties of tumors, further improving the efficacy of CDT. Therefore, the Fe/Cu-AuNP-PEG nanocomposite, with horseradish peroxidase-like, glutathione peroxidase-like, and glucose oxidase-like activities, is a promising anti-tumor agent for integrating enhanced CDT and photothermal therapy (PTT) with the enhancement of synergistic therapeutic effects.

Tannic acid-assisted fabrication of antibacterial sodium alginate-based gel beads for the multifunctional adsorption of heavy metal ions and dyes.

The existence of heavy metals and dyes seriously affects the ecological environment and human safety. Antibacterial adsorption materials with the broad-spectrum removal of multiple pollutants are urgently required for water remediation. Herein, a sustainable and antibacterial sodium alginate (SA) gel bead adsorbent with honeycomb cellular architecture is developed by the biomimetic deposition polyphenolic tannic acid (TA) induced grafting diethylenetriamine (DETA) under mild conditions for efficient removal of Cr(VI) and dyes. Taking advantage of the catechol surface chemistry, TA occurring rapid polymerization with DETA monomers not only enhances the water resistance and thermal stability of the gel bead, but also introduces abundant polyphenolic functional groups and active adsorption sites. The multifunctional gel bead showed outstanding antibacterial activity against S. aureus (sterilization rates: 83.8 %) and E. coli (sterilization rates: 99.5 %). The maximum adsorption capacity of gel bead for Cr(VI) was 163.9 mg/g. Moreover, the removal efficiency of the gel bead for dyes of Safranine T and Rhodamine B was 89.5 % (maximum adsorption capacity: 537 mg/g) and 76.7 % (maximum adsorption capacity: 460.2 mg/g), respectively, indicating its excellent broad-spectrum adsorption performance for multiple pollutants. Therefore, TA-assisted fabrication of SA-based gel bead with excellent antibacterial property is a promising multifunctional adsorption material for practical water remediation.

Functionalized photosensitive metal-organic framework as a theranostic nanoplatform for turn-on detection of MicroRNA and photodynamic therapy.

Developing a theranostic platform integrating precise diagnostic and efficient treatment is significant but challenging. Here, we reported a new theranostic platform - hairpin probe - photosensitizing MOFs (HPMOF) composed of photosensitizing MOFs (PMOFs) and hairpin probes labeled with fluorophore and quencher, in which PMOF played the role of photosensitizer and nanocarrier of the hairpin probe. The HPMOF was covered with a layer of ZIF-8 to achieve the dual-layered nanotheranostics (HPMOF@ZIF-8). The HPMOF@ZIF-8 achieved high DNA loading capacity and intracellular delivery for tumor-related miRNA imaging. Moreover, HPMOF@ZIF-8 could generate reactive oxygen species with high efficiency, which induced cell apoptosis, leading to efficient photodynamic therapy. Due to the different expression of miRNA between normal cells and cancer cells, the HPMOF@ZIF-8 could recognize cancer cells through imaging of miRNA, leading to more accurate treatment of cancer, providing a promising theranostic nanoplatform.

Noval green sodium alginate/gellan gum aerogel with 3D hierarchical porous structure for highly efficient and selective removal of Congo red from water.

Rational design of adsorbed materials with three-dimensional (3D) hierarchical porous structure, sustainable, high adsorption capacity, and excellent selective is of great significance in practical applications. Herein, a novel aerogel adsorbed material with 3D hierarchical porous architecture was fabricated by employing naturally abundant sodium alginate (SA)/gellan gum (GG) as basic construction blocks to achieve sustainability as well as applying polyethyleneimine (PEI) as functional material for highly efficient and selective capture of Congo red (CR). The aerogel sorbent exhibited strong microstructure, numerous active adsorption sites and being ultralight. The resulting aerogel adsorbent showed high adsorption capacity (3017.23 mg/g) toward CR, exceedingly most previously reported sorbents. Furthermore, the aerogel adsorbent was accompanied by outstanding selectivity for CR in four binary dye systems. Meanwhile, after 3 cycles, the adsorption capacity decreased by 14.8 %, but still maintained the adsorption capacity of 559.79 mg/g. Therefore, excellent adsorption performance, and superb selectivity prefigures its great prospects for wastewater purification.

Simultaneous detection and imaging of two specific miRNAs using DNA tetrahedron-based catalytic hairpin assembly.

Improving the accuracy, sensitivity and speed of intracellular miRNA imaging is essential for early diagnosis of cancer. To achieve this goal, we herein present a strategy for imaging two distinct miRNAs by DNA tetrahedron-based catalytic hairpin assembly (DCHA). Two nanoprobes, DTH-13 and DTH-24, were prepared by one-pot synthesis. The resultant structures were DNA tetrahedrons functionalized with two sets of CHA hairpins, which respectively responded to miR-21 and miR-155. Using these structured DNA nanoparticles as the carriers, the probes could easily enter living cells. The presence of miR-21 or miR-155 could trigger CHA between DTH-13 and DTH-24, leading to independent fluorescence signals of FAM and Cy3. In this system, the sensitivity and kinetics were significantly enhanced owing to the strategy of DCHA. The sensing performance of our method was thoroughly investigated in buffers, fetal bovine serum (FBS) solutions, living cells, and clinical tissue samples. The results validated the potential of DTH nanoprobes as a diagnostic tool for early stages of cancer.

Recent progress in the development of DNA-based biosensors integrated with hybridization chain reaction or catalytic hairpin assembly.

As isothermal, enzyme-free signal amplification strategies, hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) possess the advantages such as high amplification efficiency, excellent biocompatibility, mild reactions, and easy operation. Therefore, they have been widely applied in DNA-based biosensors for detecting small molecules, nucleic acids, and proteins. In this review, we summarize the recent progress of DNA-based sensors employing typical and advanced HCR and CHA strategies, including branched HCR or CHA, localized HCR or CHA, and cascaded reactions. In addition, the bottlenecks of implementing HCR and CHA in biosensing applications are discussed, such as high background signals, lower amplification efficiency than enzyme-assisted techniques, slow kinetics, poor stability, and internalization of DNA probes in cellular applications.