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Aflatoxin B1 (AFB1), a widespread contaminant in food and feeds, poses a threat to the health of animals and humans. Consequently, it is significant to develop a rapid, precise and highly sensitive analytical method for the detection of AFB1. Herein, we developed an immunochromatographic strip (ICS) based on a tetrahedral DNA (TDN) immunoprobe for AFB1 determination in rice bran oil. Three sizes of TDN immunoprobes (AuNP-TDN13bp-mAb, AuNP-TDN17bp-mAb, AuNP-TDN26bp-mAb) were constructed, and the performance of these three immunoprobes, including the effective antibody labeling density and immunoaffinity, was measured and compared with that of the immunoprobe (AuNP-mAb) developed using the physical adsorption method. Subsequently, the optimal TDN immunoprobe, namely AuNP-TDN13bp-mAb, was selected to prepare the immunochromatographic strip (ICS) for the qualitative and quantitative detection of AFB1 in rice bran oil. The visual limits of detection (vLODs) of the ICS based on AuNP-TDN13bp-mAb and AuNP-mAb were 0.2 ng/mL and 2 ng/mL, with scanning quantitative limits (sLOQs) of 0.13 ng/mL and 1.4 ng/mL, respectively. The ICS demonstrated a wide linear range from 0.02 ng/mL to 0.5 ng/mL, with good specificity, accuracy, precision, repeatability, and stability. Moreover, a high consistency was observed between the constructed ICS and ultra-high-performance liquid chromatography (UPLC) in the quantification of AFB1. The results indicated that the introduction of TDN was beneficial for promoting efficient antibody labeling, protecting the bioactivity of immunoprobes, and increasing the sensitivity of detection, which would provide new perspectives for the achievement of the highly sensitive detection of mycotoxins. [ABSTRACT FROM AUTHOR]

Based on the electrochemical impedance method, a marker-free biosensor with aptamer as a biometric element was developed for the determination of doxorubicin (DOX). By combining aptamer with rigid tetrahedral DNA nanostructures (TDNs) and fixing them on the surface of gold electrode (GE) as biometric elements, the density and directivity of surface nanoprobes improved, and DOX was captured with high sensitivity and specificity. DOX was captured by immobilized aptamers on the GE, which inhibited electron transfer between the GE and [Fe(CN)6]3−/4− in solution, resulting in a change in electrochemical impedance. When the DOX concentration was between 10.0 and 100.0 nM, the aptasensor showed a linear relationship with charge transfer resistance, the relative standard deviation (RSD) ranged from 3.6 to 5.9%, and the detection limit (LOD) was 3.0 nM. This technique offered a successful performance for the determination of the target analyte in serum samples with recovery in the range 97.0 to 99.6% and RSD ranged from 4.8 to 6.5%. This method displayed the advantages of fast response speed, good selectivity, and simple sensor structure and showed potential application in therapeutic drug monitoring. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1), a widespread contaminant in food and feeds, poses a threat to the health of animals and humans. Consequently, it is significant to develop a rapid, precise and highly sensitive analytical method for the detection of AFB1. Herein, we developed an immunochromatographic strip (ICS) based on a tetrahedral DNA (TDN) immunoprobe for AFB1 determination in rice bran oil. Three sizes of TDN immunoprobes (AuNP-TDN13bp-mAb, AuNP-TDN17bp-mAb, AuNP-TDN26bp-mAb) were constructed, and the performance of these three immunoprobes, including the effective antibody labeling density and immunoaffinity, was measured and compared with that of the immunoprobe (AuNP-mAb) developed using the physical adsorption method. Subsequently, the optimal TDN immunoprobe, namely AuNP-TDN13bp-mAb, was selected to prepare the immunochromatographic strip (ICS) for the qualitative and quantitative detection of AFB1 in rice bran oil. The visual limits of detection (vLODs) of the ICS based on AuNP-TDN13bp-mAb and AuNP-mAb were 0.2 ng/mL and 2 ng/mL, with scanning quantitative limits (sLOQs) of 0.13 ng/mL and 1.4 ng/mL, respectively. The ICS demonstrated a wide linear range from 0.02 ng/mL to 0.5 ng/mL, with good specificity, accuracy, precision, repeatability, and stability. Moreover, a high consistency was observed between the constructed ICS and ultra-high-performance liquid chromatography (UPLC) in the quantification of AFB1. The results indicated that the introduction of TDN was beneficial for promoting efficient antibody labeling, protecting the bioactivity of immunoprobes, and increasing the sensitivity of detection, which would provide new perspectives for the achievement of the highly sensitive detection of mycotoxins.

Shebin Hong,1– 3,* Weidong Jiang,1– 3,* Qinfeng Ding,1– 3,* Kaili Lin,1– 3 Cancan Zhao,1– 3 Xudong Wang1– 3 1Department of Oral & Cranio-Maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 2College of Stomatology, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 3National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xudong Wang; Cancan Zhao, Department of Oral and Cranio-Maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University, No. 639, Zhizaoju Road, Shanghai, 200011, People’s Republic of China, Tel +86-21-23271699, Fax +86-21-63136856, Email wang70@hotmail.com; cc.zhao@sjtu.edu.cnAbstract: Recently, programmable assembly technologies have enabled the application of DNA in the creation of new nanomaterials with unprecedented functionality. One of the most common DNA nanostructures is the tetrahedral DNA nanostructure (TDN), which has attracted great interest worldwide due to its high stability, simple assembly procedure, high predictability, perfect programmability, and excellent biocompatibility. The unique spatial structure of TDN allows it to penetrate cell membranes in abundance and regulate cellular biological properties as a natural genetic material. Previous studies have demonstrated that TDNs can regulate various cellular biological properties, including promoting cells proliferation, migration and differentiation, inhibiting cells apoptosis, as well as possessing anti-inflammation and immunomodulatory capabilities. Furthermore, functional molecules can be easily modified at the vertices of DNA tetrahedron, DNA double helix structure, DNA tetrahedral arms or DNA tetrahedral cage structure, enabling TDN to be used as a nanocarrier for a variety of biological applications, including targeted therapies, molecular diagnosis, biosensing, antibacterial treatment, antitumor strategies, and tissue regeneration. In this review, we mainly focus on the current progress of TDN-based nanomaterials for antimicrobial applications, bone and cartilage tissue repair and regeneration. The synthesis and characterization of TDN, as well as the biological merits are introduced. In addition, the challenges and prospects of TDN-based nanomaterials are also discussed.Graphical Abstract: Keywords: tetrahedral DNA nanostructure, carriers, antibacterial treatment, bone regeneration, cartilage regeneration

Modular components for rapid assembly of microfluidics must put extra effort into solving leakage and alignment problems between individual modules. Here, we demonstrate a conductive elastomer with self-healing properties and propose a modular microfluidic component configuration system that utilizes self-healing without needing external interfaces as an alternative to the traditional chip form. Specifically, dual dynamic covalent bond crosslinks (imine and borate ester bonds) established between Polyurethane (PU) and 2-Formylbenzeneboronic acid (2-FPBA) are the key to a hard room-temperature self-healing elastomeric substrate PP (PU/2-FPBA). An MG (MXene/GO) conductive network with stable layer spacing (Al-O bonds) obtained from MXene and graphene oxide (GO) by in situ reduction of metals confers photothermal conductivity to PP. One-step liquid molding obtained a standardized modular component library of puzzle shapes from PP and MGPP (MG/PP). The exosomes were used to validate the performance of the constructed microfluidic electrochemical biosensing platform. The device has a wide detection range (50–105 particles/μL) and a low limit of detection (LOD) (42 particles/μL) (S/N = 3), providing a disposable, reusable, cost-effective, and rapid analysis platform for quantitative detection of colorectal cancer exosomes. In addition, to our knowledge, this is the first exploration of self-healing conductive elastomers for a modular microfluidic electrochemical biosensing platform. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is a cytosolic DNA sensor which is regarded as a potential target for antitumor immunotherapy. However, clinical trials of STING agonists display limited anti-tumor effects and dose-dependent side-effects like inflammatory damage and cell toxicity. Here, we showed that tetrahedral DNA nanostructures (TDNs) actively enter macrophages to promote STING activation and M1 polarization in a size-dependent manner, and synergized with Mn2+ to enhance the expressions of IFN-β and iNOS, as well as the co-stimulatory molecules for antigen presentation. Moreover, to reduce the cytotoxicity of Mn2+, we constructed a TDN–MnO2 complex and found that it displayed a much higher efficacy than TDN plus Mn2+ to initiate macrophage activation and anti-tumor response both in vitro and in vivo. Together, our studies explored a novel immune activation effect of TDN in cancer therapy and its synergistic therapeutic outcomes with MnO2. These findings provide new therapeutic opportunities for cancer therapy.

Cancer patients often face severe organ toxicity caused by chemotherapy. Among these, chemotherapy‐induced hepatotoxicity and cardiotoxicity are the main causes of death of cancer patients. Chemotherapy‐induced cardiotoxicity even creates a new discipline termed "cardio‐oncology". Therefore, relieving toxicities induced by chemotherapy has become a key issue for improving the survival and quality of life in cancer patients. In this work, mesenchymal stem cell exosomes with the "G‐C" abundant tetrahedral DNA nanostructure (TDN) are modified to form a decoy exosome (Exo‐TDN). Exo‐TDN reduces DOX‐induced hepatotoxicity as the "G‐C" base pairs scavenge DOX. Furthermore, Exo‐TDN with cardiomyopathic peptide (Exo‐TDN‐PCM) is engineered for specific targeting to cardiomyocytes. Injection of Exo‐TDN‐PCM significantly reduces DOX‐induced cardiotoxicity. Interestingly, Exo‐TDN‐PCM can also promote macrophage polarization into the M2 type for tissue repair. In addition, those decoy exosomes do not affect the anticancer effects of DOX. This decoy exosome strategy serves as a promising therapy to reduce chemo‐induced toxicity. [ABSTRACT FROM AUTHOR]

A universal sensing protocol was designed for ultrasensitive fluorescence imaging of miRNAs, by combining hybridization chain reaction probe with metal nanoparticles and tetrahedral DNA nanostructure modified array chip. [Display omitted] • A universal fluorescence platform was designed to detect various target miRNAs. • Hybridization chain reaction was initiated by a programmable single-stranded DNA. • Gold nanoparticles were used to produce plasmon enhanced fluorescence. • Tetrahedral DNA nanostructure supplied antifouling property and accurate orientation. • Sensitive and accurate detection of miRNAs in complex matrix can be achieved. A versatile self-assembly probe was developed by hybridization chain reaction (HCR) with the assistance of a programmable single-stranded DNA. It addressed the issues of conventional HCR strategy that hairpin DNA sequences should be redesigned for sensing different targets, permitting convenient assay of various low-abundance targets. Combining the HCR probe with gold nanoparticles (AuNPs) and tetrahedral DNA nanostructure (TDN) modified biochip, a universal sensing protocol was proposed for ultrasensitive fluorescence imaging assay of multiple miRNAs. Benefiting from TDN and AuNPs, the biochip was endowed with the properties of accurate orientation, large specific surface area, and antifouling surface. The HCR probe was loaded with abundant Cy3 in a highly ordered way and can be captured on the biochip in the presence of target miRNA. The amount of the Cy3 on the biochip increased with the concentration of miRNA and the fluorescence signals were positively correlated with the miRNA. Impressively, the Cy3 molecules resonated with plasmons on the surface of AuNPs and amplified the fluorescence, further improving the detection sensitivity. For assay of miRNA-21, miRNA-18a, miRNA-155 and miRNA-199a, the protocol exhibited linear ranges over 4 orders of magnitude with detection limits down to 0.05 nM, 0.02 nM, 0.03 nM and 0.01 nM, respectively, high stability and throughput up to 67 tests per hour. When applied to serum samples, it also showed acceptable accuracy. The proposed protocol possessed promising potential in the diagnosis of miRNA related diseases. [ABSTRACT FROM AUTHOR]

A schematic depiction outlining the proposed mechanisms underlying the angiogenic and osteogenic effects of TDN and TDN@miR205 within the context of distraction osteogenesis (DO). [Display omitted] • A novel and effective miRNA delivery system, TDN@miRNA205, has been developed for bone healing. • A novel biological process involving TDN in bone regeneration: TDN and TDN@miR205 regulate osteogenic differentiation via vascularization. • TDN and TDN@miRNA205 activate TGF-β/BMP signaling and elevate both osteogenic and angiogenic functions. • The TDN or TDN@miRNA205 offer broad potential for Distraction osteogenesis. The clinical treatment of critical bone defects presents a great challenge, and distraction osteogenesis (DO) emerges as a highly effective strategy. However, prolonged consolidation periods resultant complications limit its clinical applicability. MicroRNA (miR) therapeutic strategies, with a notable focus on miR-205, demonstrate significant potential during DO. Nevertheless, the limited cellular uptake and facile biodegradation of miRNA constrain its applications. To address these challenges, we introduce tetrahedral DNA nanostructure (TDN), an innovative nanomaterial, engineered as gene carriers. TDN delivers the miR-205 inhibitor (TDN@miR205) to synthesize the novel nanoparticles that support bone healing for the first time. TDN@miR205 enhances the osteogenic potential of bone marrow-derived mesenchymal stem cells (BMSCs), whereas TDN alone is ineffective. Further exploration reveals TDN-modulated angiogenesis-osteogenesis communication, establishing a crucial intercellular regulatory mechanism. Notably, TDN@miR205 expedites bone formation by promoting type H vessels-related angiogenesis, secreting SLIT3 to inhibit osteoclasts, inducing SDF1 homing of BMSCs, then upregulating osteogenic genes and proteins via the TGF-β/BMP pathway, facilitating early bony ossification both in vitro and in vivo. In conclusion, TDN@miR205, the multifunctional nanoparticle-mediated vascular bone regeneration significantly contributes to ossification in DO and establishes this platform as a promising and versatile strategy for addressing DO and other complex bone diseases. [ABSTRACT FROM AUTHOR]

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring. [Display omitted] • A new Pb2+-sensing platform is constructed based on spatially localized DNA nanomachines. • The label-free detection of Pb2+ can be achieved by G-triplex/ThT fluorescent probe. • The proposed method can rapidly detect Pb2+ with a detection limit as low as 1.8 pM within 25 min • The proposed method shows good specificity and works well for Pb2+ detection in real samples. [ABSTRACT FROM AUTHOR]

Circular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. Herein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. This represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes. [Display omitted] • A DNA nanostructure-functionalized microfluidic sensor is proposed for detecting circRNA. • This biosensor can discriminate target circRNA from linear RNA counterpart and exhibits high specificity. • The chip can capture circRNA which triggers hybridization chain reaction, resulting in high detection sensitivity. • The method allows reliable detection in complex biological samples. [ABSTRACT FROM AUTHOR]

Penicillin is among the most used antibiotics for the prevention and treatment of infections in livestock. However, residual penicillin present in animal food products can cause an allergic immune response or induce several human diseases. Herein, we identified aptamer sequences highly specific to penicillin G (PenG) through GO-SELEX screening of the ssDNA library. The target-binding affinity and the dissociation constants of the aptamer sequences were determined by an electrochemical sensing platform. The effectiveness of the sensing platform was enhanced by modifying the working electrode surface using gold nanoparticles and electrochemically reduced graphene oxide and further functionalized with tetrahedral DNA nanostructures (TDNs). The TDN functionalization allowed to precisely control the surface density of the biosensing interface and orientation of attached aptamers with high target accessibility. The specificity of the selected aptamer sequence was confirmed by testing against different antibiotics. The results show that the designed aptasensor was fast, sensitive, and specific in detecting and quantifying PenG in a wide concentration range from 0.2 nM to 1 mM with an ultralow limit of detection of 0.05 nM. Additionally, the aptasensor exhibited great potential for application in a real sample when tested with penG-spiked milk samples with recovery rates in the range of 98–109%.

Stimulator of interferon genes (STING) is a cytosolic DNA sensor which is regarded as a potential target for antitumor immunotherapy. However, clinical trials of STING agonists display limited anti-tumor effects and dose-dependent side-effects like inflammatory damage and cell toxicity. Here, we showed that tetrahedral DNA nanostructures (TDNs) actively enter macrophages to promote STING activation and M1 polarization in a size-dependent manner, and synergized with Mn2+ to enhance the expressions of IFN- β and iNOS, as well as the co-stimulatory molecules for antigen presentation. Moreover, to reduce the cytotoxicity of Mn2+, we constructed a TDN–MnO 2 complex and found that it displayed a much higher efficacy than TDN plus Mn2+ to initiate macrophage activation and anti-tumor response both in vitro and in vivo. Together, our studies explored a novel immune activation effect of TDN in cancer therapy and its synergistic therapeutic outcomes with MnO 2. These findings provide new therapeutic opportunities for cancer therapy. [ABSTRACT FROM AUTHOR]

Adoptive cell therapy (ACT) is an emerging powerful cancer immunotherapy, which includes a complex process of genetic modification, stimulation and expansion. During these in vitro or ex vivo manipulation, sensitive cells are inescapability subjected to harmful external stimuli. Although a variety of cytoprotection strategies have been developed, their application on ACT remains challenging. Herein, a DNA network is constructed on cell surface by rolling circle amplification (RCA), and T cell-targeted trivalent tetrahedral DNA nanostructure is used as a rigid scaffold to achieve high-efficient and selective coating for T cells. The cytoprotective DNA network on T-cell surface makes them aggregate over time to form cell clusters, which exhibit more resistance to external stimuli and enhanced activities in human peripheral blood mononuclear cells and liver cancer organoid killing model. Overall, this work provides a novel strategy for in vitro T cell-selective protection, which has a great potential for application in ACT.

MicroRNA (miRNA) emerges as important cancer biomarker, accurate detection of miRNA plays an essential role in clinical sample analysis and disease diagnosis. However, it remains challenging to realize highly sensitive detection of low-abundance miRNA. Traditional detection methods including northern blot and real-time PCR have realized quantitative miRNA detection. However, these detection methods are involved in sophisticated operation and expensive instruments. Therefore, the development of novel sensing platform with high sensitivity and specificity for miRNA detection is urgently demanded for disease diagnosis. In this work, a novel electrochemical biosensor was constructed for miRNA detection based on target-driven cascade amplified assembly of electroactive covalent organic frameworks (COFs) on tetrahedral DNA nanostructure with multiplex recognition domains (m-TDN). COFs were employed as nanocarriers of electroactive prussian blue (PB) molecules by the "freeze-drying-reduction" method without the use of DNA as gatekeeper, which was simple, mild and efficient. The target-triggered catalytic hairpin assembly (CHA) and glutathione reduction could convert low-abundance miRNA into a large amount of Mn2+. Without the addition of exogenous Mn2+, the dynamically-generated Mn2+-powered DNAzyme cleavage process induced abundant PB-COFs probe assembled on the four recognition domains of m-TDN, resulting in significantly signal output. Using miRNA-182-5p as the model target, the proposed electrochemical biosensor achieved ultrasensitive detection of miRNA-182-5p in the range of 10 fM-100 nM with a detection limit of 2.5 fM. Taking advantages of PB-COFs probe as the enhanced signal labels, the integration of CHA, Mn2+-powered DNAzyme and m-TDN amplification strategy significantly improved the sensitivity and specificity of the biosensor. The designed sensing platform was capable of miRNA detection in complex samples, which provided a new idea for biomarker detection, holding promising potential in clinical diagnosis and disease screening. [Display omitted] • COFs acted as nanocarriers of electroactive PB, which was simple and efficient. • CHA integrated with Mn2+-powered DNAzyme achieved remarkable signal amplification. • m-TDN provided more binding sites to improve the capture efficiency of PB-COFs. • The biosensor realized miRNA-182-5p detection with a detection limit of 2.5 fM. [ABSTRACT FROM AUTHOR]

The in vitro detection of circulating tumor cells (CTCs) has been proven as a vital method for early diagnosis and evaluation of cancer metastasis, since the existence and number fluctuation of CTCs have shown close correlation with clinical outcomes. However, it remains difficult and technically challenging to realize accurate CTCs detection, due to the rarity of CTCs in the blood samples with complex components. Herein, we reported a CTCs in vitro detection strategy, utilizing a loop amplification strategy based on DNA tetrahedron and nicking endonuclease reaction, as well as the anti-background interference based on lanthanide metal luminescence strategy. In this work, a detection system (ATDN-MLLPs) composed of an aptamer-functionalized tetrahedral DNA nanostructure (ATDN) and magnetic lanthanide luminescent particles (MLLPs) was developed. ATDN targeted the tumor cells via aptamer-antigen recognition and extended three hybridizable target DNA segments from the apex of a DNA tetrahedron to pair with probe DNA on MLLPs. Then, the nicking endonuclease (Nt.BbvCI) recognized the formed double-strand DNA and nicked the probe DNA to release the target DNA for recycling, and the released TbNps served as a high signal-to-noise ratio fluorescence signal source for CTCs detection. With a detection limit of 5 cells/mL, CTCs were selectively screened throughout a linear response range of low orders of magnitude. In addition, the ATDN-MLLPs system was attempted to detect possible existence of CTCs in biological samples in vitro. [Display omitted] • A loop signal amplification strategy based on DNA tetrahedron and nicking endonuclease reaction. • Anti-Background interference based on lanthanide metal luminescence strategy. • Detection limit as low as 5 cells/mL. [ABSTRACT FROM AUTHOR]

Tetrahedral DNA nanostructure (TDN) is highly promising in developing electrochemical aptamer-based (E-AB) sensors for biomolecular detection, owing to its inherit programmability, spatial orientation and structural robustness. However, current interrogation strategies applied for TDN-based E-AB sensors, including enzyme-based amperometry, voltammetry, and electrochemical impedance spectroscopy, either require complicated probe design or suffer from limited applicability or selectivity. In this study, a TDN pendulum-empowered E-AB sensor interrogated by chronoamperometry for reagent-free and continuous monitoring of a blood clotting enzyme, thrombin, was developed. TDN pendulums with extended aptamer sequences at three vertices were immobilized on a gold electrode via a thiolated double-stranded DNA (dsDNA) at the fourth vertex, and their motion is modulated by the bonding of target thrombin to aptamers. We observed a significantly amplified signalling output on our sensor based on the TDN pendulum compared to E-AB sensors modified with linear pendulums. Moreover, our sensor achieved highly selective and rapidly responsive measurement of thrombin in both PBS and artificial urine, with a wide dynamic range from 1 pM to 10 nM. This study shows chronoamperometry-enabled continuous biomarker monitoring on a sub-second timescale with a drift-free baseline, demonstrating a novel approach to accurately detect molecular dynamics in real time. [Display omitted] • A Tetrahedral DNA Nanostructure (TDN) pendulum for continous biomarker monitoring. • Enhancement in signal outputs compared to linear pendulum sensors. • Chronamperometry interrogation with millisecond resolution was achieved. • Quantative and selective measurements of thrombin ranging from 1 pM to 10 nM. • Real-time monitoring of thrombin in a reagent-free manner was demonstrated. [ABSTRACT FROM AUTHOR]

Modular components for rapid assembly of microfluidics must put extra effort into solving leakage and alignment problems between individual modules. Here, we demonstrate a conductive elastomer with self-healing properties and propose a modular microfluidic component configuration system that utilizes self-healing without needing external interfaces as an alternative to the traditional chip form. Specifically, dual dynamic covalent bond crosslinks (imine and borate ester bonds) established between Polyurethane (PU) and 2-Formylbenzeneboronic acid (2-FPBA) are the key to a hard room-temperature self-healing elastomeric substrate PP (PU/2-FPBA). An MG (MXene/GO) conductive network with stable layer spacing (Al-O bonds) obtained from MXene and graphene oxide (GO) by in situ reduction of metals confers photothermal conductivity to PP. One-step liquid molding obtained a standardized modular component library of puzzle shapes from PP and MGPP (MG/PP). The exosomes were used to validate the performance of the constructed microfluidic electrochemical biosensing platform. The device has a wide detection range (50–105 particles/μL) and a low limit of detection (LOD) (42 particles/μL) (S/N = 3), providing a disposable, reusable, cost-effective, and rapid analysis platform for quantitative detection of colorectal cancer exosomes. In addition, to our knowledge, this is the first exploration of self-healing conductive elastomers for a modular microfluidic electrochemical biosensing platform.

Adoptive cell therapy (ACT) is an emerging powerful cancer immunotherapy, which includes a complex process of genetic modification, stimulation and expansion. During these in vitro or ex vivo manipulation, sensitive cells are inescapability subjected to harmful external stimuli. Although a variety of cytoprotection strategies have been developed, their application on ACT remains challenging. Herein, a DNA network is constructed on cell surface by rolling circle amplification (RCA), and T cell-targeted trivalent tetrahedral DNA nanostructure is used as a rigid scaffold to achieve high-efficient and selective coating for T cells. The cytoprotective DNA network on T-cell surface makes them aggregate over time to form cell clusters, which exhibit more resistance to external stimuli and enhanced activities in human peripheral blood mononuclear cells and liver cancer organoid killing model. Overall, this work provides a novel strategy for in vitro T cell-selective protection, which has a great potential for application in ACT. T cell-selective coating and protection strategy with a flexible DNA network was formed by rolling circle amplification, which promotes T-cell aggregation and activation for anti-tumor therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

This study introduces an innovative detection system for multiple cancer biomarkers, employing transcription isothermal amplification methods in conjunction with a tetrahedral DNA nanostructure (TDN). We demonstrate that TDN enhances various transcription isothermal amplification methods by placing DNA probes in proximity. Notably, the TDN-enhanced split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR) system stands out for its optimal performance and operational simplicity, especially in identifying non-coding RNAs such as microRNAs and long non-coding RNAs (lncRNAs). Multiplex detection of lncRNAs was also achieved by generating distinct light-up RNA aptamers, each emitting unique fluorescence signals. The system effectively identified the target lncRNAs, demonstrating high sensitivity and selectivity in both cell lines and clinical samples. The system, utilizing the single enzyme T7 RNA polymerase, can be easily tailored for alternative targets by substituting target-specific sequences in DNA probes and seamlessly integrated with other isothermal amplification methods for greater sensitivity and accuracy in the detection of multiple cancer biomarkers. • TDN enhances various transcription-based isothermal amplification methods. • TDN-enhanced STAR stands out for its optimal performance and operational simplicity. • Multiplex detection of lncRNAs was achieved with high sensitivity and specificity. • The target lncRNAs were effectively identified in cell lines and clinical samples. [ABSTRACT FROM AUTHOR]

A multi-amplified electrochemiluminescence (ECL) aptasensor was developed for efficient recognition of CA125 using tetrahedral DNA nanostructure (TDN) enhanced toehold-mediated strand displacement (TMSD) coupled with gold nanoparticles/Ru(bpy) 3 2+/metal–organic framework (AuNPs/Ru/ZIF-MOF) signal probe. [Display omitted] • TMSD was utilized to improve the sensitivity of the sensor. • TDN was used to enhance the efficiency of TMSD amplification. • ZIF-MOF improved the immobilizing amount of Ru(bpy) 3 2+ and gold nanoparticles. • Detection limit of 6 × 10-3 pg/mL obtained for CA125 based on TDN based TMSD. • The assay achieved the detection of CA125 in human serum samples. Cancer antigen 125 (CA125) is a typical tumor marker of ovarian cancer. Here, a multi-amplified electrochemiluminescence (ECL) aptasensor was developed for efficient recognition of CA125 using tetrahedral DNA nanostructure (TDN) enhanced toehold-mediated strand displacement (TMSD) coupled with gold nanoparticles/Ru(bpy) 3 2+/metal–organic framework (AuNPs/Ru/ZIF-MOF) signal probe. AuNPs and Ru(bpy) 3 2+ modified ZIF-MOF acted as initial ECL signal and further used for the immobilization of TDN, the activated DNA templates on the surface of TDN were firstly hybridized with ferrocene labeled DNA probe (S6) and S5, in which, S6 acted as the energy acceptor of ECL signal from Ru(bpy) 3 2+, making the sensor in a "signal-off" state. After the specific recognition of aptamer (AP) with CA125, DNA initiator (S7) was freed to induce the happen of TMSD by using S8 as the helper DNA, accompanying with the release of S6 from the electrode surface and the recovery of Ru(bpy) 3 2+ ECL signal, making the sensor in a "signal-on" state. Then, S7 was recycled for the next TMSD, making the sensor highly sensitive with a detection limit of 6 × 10-3 pg/mL. Moreover, the proposed aptasensor achieved high performance for CA125 detection in human serum samples, illustrating the reliability of the sensor in clinical analysis. [ABSTRACT FROM AUTHOR]

In this work, we proposed an electrochemical aptasensor for patulin (PAT) based on tetrahedral DNA nanostructures (TDNs) and thionine (Thi)-labeled Fe 3 O 4 nanoparticles (Fe 3 O 4 NPs)/rGO signal amplification strategy. The rigid structure of TDNs could effectively improve the binding efficiency. Fe 3 O 4 NPs/rGO with excellent electrical conductivity and large specific surface area was used as a label material, which could load more Thi and accelerate electron transfer. Besides, the unique catalytic properties of Fe 3 O 4 NPs could achieve active signal amplification. Once PAT existed, PAT aptamer was released from the capture probe, thereby introducing Fe 3 O 4 NPs/rGO with Thi onto the electrode surface. Therefore, a noticeable increase in Thi current intensity was observed. Under the optimized conditions, the proposed aptasensor showed superior performance with a linear range from 5 × 10−8 to 5 × 10−1 μg mL−1 and a detection limit of 30.4 fg mL−1. The obtained sensor showed reliable specificity, stability and reproducibility, and was successfully applied to the determination of real samples. A tetrahedral DNA nanostructures (TDNs)-based electrochemical aptasensor was successfully constructed for detection of patulin (PAT) using the Fe 3 O 4 nanoparticles (Fe 3 O 4 NPs)/rGO for Signal Amplification of thionine (Thi). Image 1 • A novel TDNs-based electrochemical aptasensor for the detection of patulin was first constructed. • Fe 3 O 4 NPs/rGO successfully acted as a fix substrate for Thi and promoted the electron transfer between Thi and Au electrode. • The fabricated immunosensor exhibited satisfactory specificity, good stability and excellent reproducibility. [ABSTRACT FROM AUTHOR]

Outbreaks of infectious viruses cause enormous challenges to global public health. Recently, the coronavirus disease 2019 (COVID-19) induced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely threatened human health and resulted in the global pandemic. A strategy to detect SARS-CoV-2 with both fast sensing speed and high accuracy is urgently required. Here, rapid detection of SARS-CoV-2 antigen using carbon-nanotube-array-based thin-film transistor (CNT-array-based TFT) biosensors merged with tetrahedral DNA nanostructures (TDNs) and triple aptamers is demonstrated for the first time. Compared with CNT-network-based TFT biosensors and metal-electrode-based CNT-TFT biosensors, the response of CNT-array-based TFT biosensors can be enhanced up to 102% for SARS-CoV-2 receptor-binding domain (RBD) detection, which is supported by its sensing mechanism. By combining TDNs with triple aptamers, the biosensor has realized the wildtype SARS-CoV-2 RBD detection in a broad detection range spanning eight orders of magnitude with a low limit of detection (LOD) of 10 aM (6 copies/μL) owing to the improved protein capture efficiency. Moreover, the triple-aptamer biosensor platform has achieved the detection of SARS-CoV-2 Omicron RBD in a low LOD of 6 aM (3.6 copies/μL). Additionally, the CNT-array-based TFT biosensors have exhibited excellent specificity, enabling identification among SARS-CoV-2 antigen, SARS-CoV antigen and MERS-CoV antigen. The platform of CNT-array-based TFT biosensors combined with TDNs and triple aptamers provides a high-performance and rapid approach for SARS-CoV-2 detection, and its versatility by altering specific aptamers enables the possibility for rapid virus detection. [Display omitted] • A CNT-array-based thin-film transistor for SARS-CoV-2 detection is reported. • The biosensor response can improve to 102% using triple-aptamer DNA tetrahedrons. • The detection of wildtype SARS-CoV-2 RBD was achieved in a low LOD of 10 aM. • The platform realized the SARS-CoV-2 Omicron RBD detection with LOD of 6 aM. • The distinction among antigens of SARS-CoV-2, SARS-CoV and MERS-CoV was achieved. [ABSTRACT FROM AUTHOR]

Here we develop robust noncovalent spherical nucleic acid enzymes (SNAzymes) for direct electrochemiluminescence (ECL) detection of acute myocardial infarction (AMI) related endogenous microRNAs in both circulating blood and cardiomyocytes, which circumvents the need for time-consuming signal amplification widely used in previous counterparts. It mainly relies on the super peroxidase-like activity of the designed noncovalent SNAzymes, promoted by a few nucleotides flanking on the 3′-terminals of common parallel G-quadruplexes (G4). For this reason, an unmodified G4 with an A 5 T 30 head is well chosen and then attached robustly onto bare AuNPs via microwave-assisted heating-drying. A probe strand is meanwhile attached onto SNAzymes, enabling the target microRNA-triggered formation of a Y-shaped junction together with a capture strand tethered to a DNA tetrahedron on the electrode surface. The utilization of this tetrahedral nanoscaffold favors the ECL readout and thereby contributes to high sensitivity of the sensing platform. In this way, an AMI-related microRNA, miR-499, can be probed in a wide linear range, with a detection limit of 33 aM and high selectivity over other analogues. Furthermore, our developed sensing platform is employed to analyze endogenous miR-499 in AMI patients' blood, revealing an apparently higher level than the mean value of the healthy. What it means to patients, heart injury, is elucidated by comparing the miR-499 levels of cardiomyocytes and other tissue cells, with endogenous miR-16 as an intrinsic reference. [Display omitted] [ABSTRACT FROM AUTHOR]

Large-sized DNA structures have been extensively explored in electrochemical sensing for abundant binding sites or large steric hindrance, yet large steric hindrance could hinder electron transfer of probes on DNA, depressing their efficiency for signal amplification. Herein, a ratiometric electrochemical aptasensor was designed to take advantage of abundant binding sites and large steric hindrance of the tetrahedral DNA nanostructure (TDN) for efficient signal amplification. The working principle is that the recognition of the target leads to a decrease in binding sites and a reduction in steric hindrance, which in turn leads to the opposite change in the two signals. Ferrocene (Fc)-labeled complementary DNA acted as a substrate, while a TDN containing aptamer was used for fumonisin B1 (FB1) recognition and methylene blue (MB) absorption. The binding of FB1 triggered the stripping of TDN, leading to a decreased current of MB (I MB) and an increased current of Fc (I Fc). In this way, TDN allowed the enlarged variations in I Fc and I MB by the significant change in steric hindrance and binding sites in ratiometric electrochemical sensing, respectively. Consequently, the developed aptasensor offered a linear range of 0.1–100 pg mL−1 and a detection limit of 0.087 pg mL−1 for FB1. This strategy offers a new way to fabricate high-performance sensors with large DNA structures. • A ratiometric electrochemical aptasensor was designed with signal amplification. • TDN with abundant binding sites and large steric hindrance was introduced. • The stripping of TDN led to the opposite change of two signals. • The ratiometric aptasensor showed high analytical performance for FB1 detection. [ABSTRACT FROM AUTHOR]

Complications arising from diabetes can threaten multiple organs. Advanced glycation end products (AGEs) play a significant role in inducing these complications. Highly processed diets and hyperglycemia facilitate the accumulation of AGEs in the body. Interaction between AGEs and their main receptor (RAGE) initiates the transmission of intracellular inflammatory and cell death signals, which ultimately lead to complications. To counter AGEs-induced damage, we developed an siRNA-binding tetrahedral framework nucleic acids (TDN) system, termed Tsi, which combines the potent cell membrane penetrability and serum stability of TDN with the gene-targeting specificity of siRNA-RAGE. Tsi effectively and persistently downregulates the expression of RAGE, thereby suppressing inflammation by blocking the NF-κB pathway as well as exhibiting antioxidant functions. Furthermore, Tsi regulates the pyroptosis state of macrophages via the NLRP3/caspase-1 axis, which inhibits the spread of cell death signals and maintains homeostasis. This is of great significance for the synergistic treatment strategy for systemic complications in patients with refractory hyperglycemia. In summary, this study describes a nanomedicine that targets the RAGE and suppresses AGE-induced inflammation. This nucleic acid drug holds long-lasting efficacy and is independent of lowering hyperglycemia, which provides a strategy for the treatment of diabetic complications and age-related diseases.

Ultrasensitive identification of biomarkers in biofluids is essential for the precise diagnosis of diseases. For the gold standard approaches, polymerase chain reaction and enzyme-linked immunosorbent assay, cumbersome operational steps hinder their point-of-care applications. Here, a bionic biomarker entrapment system (BioES) is implemented, which employs a multi-body Y-shaped tetrahedral DNA probe immobilized on carbon nanotube transistors. Clinical identification of endometriosis is successfully realized by detecting an estrogen receptor, ERβ, from the lesion tissue of endometriosis patients and establishing a standard diagnosis procedure. The multi-body Y-shaped BioES achieves a theoretical limit of detection (LoD) of 6.74 aM and a limit of quantification of 141 aM in a complex protein milieu. Furthermore, the BioES is optimized into a multi-site recognition module for enhanced binding efficiency, realizing the first identification of monkeypox virus antigen A35R and unamplified detection of circulating tumor DNA of breast cancer in serum. The rigid and compact probe framework with synergy effect enables the BioES to target A35R and DNA with a LoD down to 991 and 0.21 aM, respectively. Owing to its versatility for proteins and nucleic acids as well as ease of manipulation and ultra-sensitivity, the BioES can be leveraged as an all-encompassing tool for population-wide screening of epidemics and clinical disease diagnosis., (© 2023 Wiley-VCH GmbH.)

Objective: To explore the effects and underlying biological mechanisms of tetrahedral DNA nanostructures (TDNs) on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Materials and methods: Real‐time cell analysis (RTCA) and CCK8 were used to screen the best concentration of TDN for PDLSCs. Cell proliferation and osteogenic differentiation were assessed after PDLSCs were treated with TDN. Data were analysed using one‐way ANOVA. Results: Tetrahedral DNA nanostructures could play a crucial role in accelerating the proliferation of PDLSCs and had the strongest promotive effect on PDLSCs at a concentration of 250 nmol/L. Simultaneously, the osteogenic differentiation of PDLSCs could be promoted significantly by TDNs and the finding displayed that the Wnt/β‐catenin signalling pathway might be the underlying biological mechanisms of TDNs on promoting the osteogenic differentiation of PDLSCs. Conclusion: Tetrahedral DNA nanostructure treatment facilitated the proliferation of PDLSCs, significantly promoted osteogenic differentiation by regulating the Wnt/β‐catenin signalling pathway. Therefore, TDNs could be a novel nanomaterial with great potential for application to PDLSC‐based bone tissue engineering. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract In recent years, there has been a rapid progress in the development of DNA nanomaterials for biological applications. Owing to their unsurpassed editability, various DNA nanomaterials with well-documented biocompatibility and relative stability have been developed. Among these tetrahedral DNA nanostructures (TDNs), as typical multi-arm DNA tiles for 3D polyhedral, is a major research focus owing to their structural and biological features. TDNs have yielded satisfactory results as biological regulators, biosensors, and targeted delivery vehicles. Extensive studies on TDNs have revealed that TDNs alone could regulate several cellular processes, such as migration, proliferation, differentiation, and autophagy. Further, their editability enables control of the spatial distribution of biosensors, thereby promoting accuracy and efficiency. Finally, various modification procedures allow attachment of nucleic acids, aptamers, peptides, antibodies, and various low-molecular-weight drugs to the TDNs, thereby yielding a novel targeted delivery system. This review summarizes recent research progress on TDNs in these aforementioned major areas. These discoveries might be of great therapeutic value in regenerative medicine, gene delivery, targeted chemotherapy, and other related fields. We also illustrate the emerging challenges in the clinical application of TDNs, as well as future development opportunities. [ABSTRACT FROM AUTHOR]

Abstract Here, we report an electrochemical sensor for rapid and sensitive detection of octamer-binding transcription factor 4 (Oct4) in human tissue samples by utilizing a designed tetrahedral DNA nanostructure (TDN). In the design, the TDN is also extended with two additional strands from two vertices. When Oct4 is absent, the strands are linked together by complementary pairing bases. Owing to the rigid structure of TDN, contact of the redox labels on the signal strand and electrode surface is greatly prohibited, resulting in a lower electrochemical signal. However, the specific binding of Oct4 to the edge of the tetrahedron will liberate the signal strand and increase the redox current dramatically. Experimental results reveal that the proposed sensor shows a linear range of 0.5–1000 ng/mL with a detection limit of 60 pg/mL. Moreover, it can be directly applied to clinical sample detection. This sensor can also achieve one-step detection of Oct4 in less than 30 min. Furthermore, through replacing the binding site, this sensor can be easily extended to a wide application range of DNA binding proteins. Highlights • A novel electrochemical sensor for Oct4 assay is developed. • The sensor is based on target-induced steric hindrance effect on a tetrahedral DNA nanostructure. • Oct4 is detected effectively in real clinical tissue samples. • The sensor may have great potential for the diagnosis of cancer. [ABSTRACT FROM AUTHOR]

Dental pulp stem cells (DPSCs) derived from the human dental pulp tissue have multiple differentiation capabilities, such as osteo/odontogenic differentiation. Therefore, DPSCs are deemed as ideal stem cell sources for tissue regeneration. As new nanomaterials based on DNA, tetrahedral DNA nanostructures (TDNs) have tremendous potential for biomedical applications. Here, the authors aimed to explore the part played by TDNs in proliferation and osteo/odontogenic differentiation of DPSCs, and attempted to investigate if these cellular responses could be driven by activating the canonical Notch signaling pathway. Upon exposure to TDNs, proliferation and osteo/odontogenic differentiation of DPSCs were dramatically enhanced, accompanied by up regulation of Notch signaling. In general, our study suggested that TDNs can significantly promote proliferation and osteo/odontogenic differentiation of DPSCs, and this remarkable discovery can be applied in tissue engineering and regenerative medicine to develop a significant and novel method for bone and dental tissue regeneration. [ABSTRACT FROM AUTHOR]

Rapid and accurate detection of Vibrio parahaemolyticus (VP) in a variety of testing environments is critical for preventing the spread of foodborne illnesses. Therefore, a dual-mode colorimetric-electrochemical biosensor was prepared for the rapid and sensitive detection of VP. Phenylboronic acid-decorated CuO 2 nanodot-mediated organic metal-organic framework (CP@MOF) nanozymes were constructed to produce colorimetric and electrochemical signals. Furthermore, an electrode surface was fabricated using tetrahedral DNA nanostructure-functionalized poly(amidoamine) dendrimer-Au nanoparticles to specifically bind VP. Under acidic environments, the CP@MOF nanozymes disintegrated, enabling the simultaneous release of Cu2+ and H 2 O 2. This in turn causes a Fenton-type reaction, generating a hydroxyl radical, which then oxidizes 3,3′,5,5′-tetramethylbenzidine, thereby enabling visual detection of VP. Moreover, the produced copper ions can be used for the electrochemical detection of VP. This dual-mode biosensor achieved an accuracy of 88.7 %, which was higher than that of single-mode assays (81.1 %). Hence, this dual-mode assay may facilitate the effective on-site detection of pathogens in shrimp samples. [Display omitted] • A CP@MOF nanozyme-based dual-mode biosensor was made to detect VP. • The dual-mode biosensor features generate both visual and electrochemical signals. • The PBA-CP@MOF/ VP /the electrode sandwich structures was formed to emit signals. • The biosensor provides high specificity and sensitive detection for VP. • This dual-mode biosensor achieved an accuracy of 88.7 %. [ABSTRACT FROM AUTHOR]

It is well-established that the combined use of nanostructured substrates and immunoaffinity agents can enhance the cell-capture performance of the substrates, thus offering a practical solution to effectively capture circulating tumor cells (CTCs) in peripheral blood. Developing along this strategy, this study first demonstrated a top-down approach for the fabrication of tetrahedral DNA nanostructure (TDN)-NanoGold substrates through the hierarchical integration of three functional constituents at various length-scales: a macroscale glass slide, sub-microscale self-organized NanoGold, and nanoscale self-assembled TDN. The TDN-NanoGold substrates were then assembled with microfluidic chaotic mixers to give TDN-NanoGold Click Chips. In conjunction with the use of copper (Cu)-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated CTC capture and restriction enzyme-triggered CTC release, TDN-NanoGold Click Chips allow for effective enumeration and purification of CTCs with intact cell morphologies and preserved molecular integrity. To evaluate the clinical utility of TDN-NanoGold Click Chips, we used these devices to isolate and purify CTCs from patients with human papillomavirus (HPV)-positive (+) head and neck squamous cell carcinoma (HNSCC). The purified HPV(+) HNSCC CTCs were then subjected to RT-ddPCR testing, allowing for detection of E6/E7 oncogenes, the characteristic molecular signatures of HPV(+) HNSCC. We found that the resulting HPV(+) HNSCC CTC counts and E6/E7 transcript copy numbers are correlated with the treatment responses in the patients, suggesting the potential clinical utility of TDN-NanoGold Click Chips for noninvasive diagnostic applications of HPV(+) HNSCC. [Display omitted] • Construction of tetrahedral DNA nanostructure-NanoGold substrate for purifying CTCs. • TDN-NanoGold Click Chip combined CuAAC and restriction enzyme for CTC purification. • HPV E6/E7 oncogenes were detected in CTCs purified from HPV(+) HNSCC patients. • TDN-NanoGold Click Chip can be used for noninvasive diagnosis of HPV(+) HNSCC. [ABSTRACT FROM AUTHOR]

Oxidative damage is an important factor in causing various human disease and injury. As an oxidative DNA damage product, 8-hydroxy-2′-deoxyguanosine (8-OHdG) is a key marker, which is widely used to study oxidative damage mechanism in diseases. Most reported electrochemical methods were based on oxidation current of 8-OHdG. In this work, a simple electrochemical biosensor for ultrasensitive detection of 8-OHdG was proposed based on it triggered polyaniline (PANI) deposition on tetrahedral DNA nanostructure (TDN). TDN was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences. 8-OHdG-aptamer on the top of TDN formed a hemin/G-quadruplex structure in the presence of 8-OHdG and hemin, which have high catalytic activity to trigger PANI deposition. Numerous negative charges on the duplex DNAs contained in hemin/G-quadruplex and TDN supplied exquisite environment for PANI deposition, which improved the detection sensitivity greatly by increasing the DPV current to10-fold (∼3 μA) compared to our previously reported method without TDN. The response signals correlated linearly with the concentration of 8-OHdG ranging from 10 pM to 2 nM, with a detection limit of 1 pM (S/N = 3). The sensitivity was improved to almost 300-fold when compared with most of previously reported electrochemical methods. The method was also simple and reliable, avoiding complex, expensive label procedures and nanomaterial synthesized procedures. The method had been successfully applied to quantify 8-OHdG in urine and human serum samples with satisfactory results. [ABSTRACT FROM AUTHOR]

[Display omitted] • A simple and one-pot fluorescence platform was constructed for detecting T4 PNK activity. • The introduction of TDN endowed this strategy with improved amplification efficiency and homogeneous experimental conditions. • The tandem repeated G4 sequence generated by PER exhibited strong fluorescence enhancement. • The detection limit of 1.8 × 10−5 U/mL was realized by this strategy. • The practicality of this strategy was verified in the detection human serum and cell lysates. T4 polynucleotide kinase (T4 PNK), which acts as a bifunctional enzyme, is widely involved in important phosphorylation and dephosphorylation processes. Sensitive and specific detection of T4 PNK activity will help to elucidate its critical importance in disease diagnosis and treatment. The conventional detection method uses hairpin or single-strand primers modified with phosphorylation group as the substrates for T4 PNK hydrolysis. However, considering to the large spatial distances and disordered reaction orientation among the substrates and enzymes, the efficiency and sensitivity of the method are somewhat limited. Here, we propose a simple and sensitive signal amplification strategy, integrating the tetrahedral DNA nanostructure with primer exchange reaction (TDN-PER), which ensures different reaction direction and improves the local reaction concentrations. Compared to dispersed single PER, localized TDN-PER provides more primers for initiating signal amplification. Benefiting from the unique structural transformation from dispersion to localization, this strategy provided ultrahigh sensitivity, achieving a detection limit of 1.8 × 10−5 U/mL, while maintaining excellent accuracy and specificity in complex human serum and cell lysates environments. The method successfully evaluated the inhibitory effects of (NH 4) 2 SO 4 and Na 2 HPO 4 on T4 PNK activity. Finally, the practical application capability of this strategy was verified in the detection of cell-free extracts, providing a promising analytical platform for T4 PNK-related biological and medical research. [ABSTRACT FROM AUTHOR]

A novel tetrahedral DNA nanostructure (TDN) functionalized paper-based colorimetric sensor was developed for ultrasensitive analysis of mercury ion (Hg2+). On the paper-based platform, TDN was used as scaffold to anchor aptamer to improve the capture efficiency owing to the controllably specific orientation, low steric hindrance effect and well-defined spacing. In the absence of Hg2+, biotinylated aptamer would bind with streptavidin-labeled HRP. Whereas, in the presence of Hg2+, aptamer would preferentially bind to Hg2+ owing to the higher binding constant of T–Hg–T than that of T–A, which consequently leading to the biotinylated aptamer releasing from TDN. After introducing TMB-H 2 O 2 solution, strong variation of color signal could be observed. Therefore, the quantitative analysis of Hg2+ was carried out by using smartphone to monitor the color intensity on paper chip. The proposed strategy exhibits good linearity over a wide range of 10–13–10–7 M with a low detection limit of 30 fM. And this method had been successfully applied in real water samples. Our strategy provided a versatile, low-cost and user-friendly paper-based monitoring platform for rapid and ultrasensitive analysis of heavy metal ions and thus had potential applications in food safety and public health. [Display omitted] • A sensitive paper-based platform based on tetrahedral DNA nanostructure. • DNA nanostructure confers controllable anchoring aptamer in a consistently specific orientation and well-defined spacing. • Employment of high affinity of aptamer toward target mercury ion with 30 fM detection limit. • The developed platform is simplicity, rapidity, user-friendly, low cost and sensitive monitor food quality. [ABSTRACT FROM AUTHOR]

Antibiotics are widely used for improving the living conditions of livestock. However, residual antibiotics present in animal products induce several human diseases. Therefore, a simple, rapid, and cost-effective system for detecting and monitoring the presence of antibiotics in foods is in great demand to alleviate safety concerns. In this study, a highly sensitive and selective aptameric electrochemical sensing platform was designed based on nanomaterial modification and DNA nanotechnology. Electrochemically reduced graphene oxide and gold nanoparticles were used to modify the working surface of a screen-printed electrode to enhance electrical conductivity and biocompatibility. The electrode surface was further modified with self-assembled tetrahedral DNA nanostructures (TDN) to improve the detection sensitivity. The TDN allowed controlling the nano-spacing of aptamers immobilized on the electrode surface and placing aptamers in a solution-phase-like detecting environment to improve the target-binding efficiency without signal amplification modules. Differential pulse voltammetry was employed to measure electrical signals in proportion to the amount of ampicillin, the target antibiotic, present in buffer and spiked milk samples. The designed aptasensor was able to detect and measure the target ampicillin in less than 30 min over a wide concentration range of 10 pM to 1 mM, with a limit of detection of 1 pM, which is 100 times better than when using the same sensing probe without TDN modification. The aptasensor was reusable by simply rinsing with deionized water, remained stable during 15-day storage, and yielded reproducible results. The detection of antibiotic ampicillin using TDN- and nanomaterials-modified electrode. [Display omitted] • An electrochemical aptasensor was designed based on tetrahedral DNA (TDN) nanostructure. • The TDN help to control aptamer orientation and reduces the steric hindrance effect and enhance the detection sensitivity. • The LOD and linear range of the aptasensor are 1 pM and 10 pM to 1000 nM of ampicillin, respectively. • The recovery rate of ampicillin in real milk samples is 97–104%. • The aptasensor has excellent regeneration capacity and reproducibility. [ABSTRACT FROM AUTHOR]

We study the capture and translocation characteristics of DNA tetrahedron with varying degrees of assembly to evaluate their self-assembly process and the potential of big-sized tetrahedral DNA nanostructures as a surrogate label for biomarkers of interest, designed for fast and reliable specific detection based on conductance blockages. [Display omitted] • The capture and translocation characteristics of DNA tetrahedron with varying degrees of assembly were studied to evaluate their self-assembly process. • The nanopipette sensing for small molecular assisted by big-sized tetrahedral DNA nanostructure is simple and efficient. • The present study proved that single small molecule transport is quite possibly be detected using large scale nanopore with assisting of tetrahedral DNA nanostructure. • The combination of nucleic acid aptamer and tetrahedral DNA nanostructure realizes specific sensing of small molecules by nanopipette. Nanopipette as a member of solid-state nanopores has been developed to detect a set of biological molecules. Although the size of the nanopipette can be adjusted artificially, the direct detection of small molecules is still challenging, which is limited by the precision of the nanopipette preparation. In this work, the translocation characteristics of DNA tetrahedrons with varying degrees of the assembly were studied to evaluate their self-assembly process and the advantage of tetrahedral DNA nanostructures as a signal amplification label for biomarkers detection based on current blockages. Self-assembly of different number of single-stranded DNA causes the DNA nanostructure in different shapes. Through the complete assembly of DNA tetrahedron, we demonstrate that the nanopore signal-to-noise ratio is significantly increased and the event signal becomes more stable and significant. Therefore, these attributes allow us to gain more insight into the assembly process of DNA nanostructures and we can use tetrahedral DNA nanostructure to develop an effective assay for identifying small molecules with the nanopipette. [ABSTRACT FROM AUTHOR]

• Tetrahedral DNA nanostructure provides an effective drug delivery vehicle. • We constructed a 3D-bioprinted scaffold loaded with TDN-clindamycin complex. • The novel scaffold was biocompatible, with osteogenic and antimicrobial properties. • The scaffold significantly improved repair in rat model of infected bone defect. Infection of bone defects is a common clinical problem that negatively impacts bone repair and may result in the development of antibiotic resistance due to the long-term use of antibiotics. To the best of our knowledge, integrating tetrahedral DNA nanostructure (TDN) drug delivery with 3D bioprinting has not been reported to treat infected bone defects. However, numerous studies have focused on effectively controlling infected bone defects and achieving functional reconstruction. In this study, TDN was proposed as a drug delivery vehicle to enhance cell penetration and the antibacterial properties of clindamycin (CLI), a common antibiotic used to treat osteomyelitis. A 3D hybrid scaffold loaded with TDN-CLI complexes was constructed using bioprinting technology. Its structure and biological properties were characterized, and the scaffold was further applied to treat methicillin-resistant Staphylococcus aureus (MRSA) infection in a rat model of bone defect. The results demonstrated that the TDN-CLI-loaded 3D bioprinted hybrid scaffold possessed excellent biocompatibility, outstanding osteogenic and antimicrobial activity, and significantly improved the repair of infected bone defects in the rat model. The TDN-CLI-loaded hybrid scaffold developed in the current study has broad application prospects for treating infected bone defects. [ABSTRACT FROM AUTHOR]

Fumonisin B1 (FB1) is common contaminant in agricultural and food products, and sensors with high sensitivity and broad dynamic range are extremely required for FB1 determination. Herein, we reported the tetrahedral DNA nanostructure (TDN)-enabled electrochemical aptasensor for FB1 detection at sub-fg mL−1 level with a dynamic range of 7-folds magnitude. TDN with side length of 5.67 nm was designed to fabricate the substrate for FB1 aptamer anchoring, aiming to achieve the controllable and stable assembly of sensing interface by tuning the assembly density of TDN; TDN at the electrode acts as the anchor for aptamer, absorption of methylene blue as signal generator. Assistant aptamer was added into the sample solution to deplete FB1, reducing the amount of free FB1, thereby extending dynamic range of sensing system. Simultaneously, the negatively-charged TDN can efficiently depress absorption of free aptamer in sample solution. In this way, the utilization of TDN helps to enable the sensor with higher sensitivity and extended dynamic range for FB1. Consequently, the developed sensor allowed a linear dynamic range of 0.500 fg mL−1~1.00 ng mL−1, while the limit of detection was offered to be 0.306 fg mL−1, which was the lowest among the available methods. The applicability of aptasensor was validated by rice sample analysis, which exhibited the comparable reliability of liquid-chromatography tandem mass spectrometry/mass spectrometry. Our work has provided an efficient sensing platform for FB1, and the strategy can be also extended to fabricate sensors for other mycotoxins. • TDN was applied to controllable assembly of sensing interface, which endowed aptasensor with the lowest LOD among all reported sensors for FB1. • Negatively-charged TDN was subtly applied to generate electrostatic repulsion on assistant aptamer, addressing the impact of interfering substances. • TDN was smartly adsorbed methylene blue to generate large and stable redox current. [ABSTRACT FROM AUTHOR]

Adopting carbon nanotube (CNT) transistors as biosensors has been developed as a promising method for cancer biomarker detection, which has shown superior sensitivity and selectivity. However, the detection of circulating tumor DNA (ctDNA) by the CNT transistor based biosensors is still a challenge and no work has been reported. Here, direct label-free DNA detection of AKT2 gene related to triple-negative breast cancer by all-CNT thin-film transistor (TFT) biosensors incorporated with tetrahedral DNA nanostructures (TDNs) is proposed and achieved for the first time. The adoption of TDNs enables improved biosensor response for at least 35% and even as high as 98% as compared with single-stranded DNA (ssDNA) probes owing to the enhanced DNA hybridization efficiency. Influence of the TDNs' linker length on the biosensor performance is important and has been investigated. Concentration-dependent DNA detection is achieved by the all-CNT TFT biosensors with a broad linear detection range of six orders of magnitude and a theoretical limit of detection (LOD) of 2 fM. In addition, the all-CNT TFT biosensors exhibit favorable selectivity and repeatability. The platform of all-CNT TFT biosensors incorporated with TDNs has great potential for multiplexed detection of various cancer biomarkers, providing a simple yet high performance universal strategy for low-cost clinical applications. • A tetrahedral DNA nanostructure (TDN) based all-carbon nanotube thin-film transistor biosensor was developed. • The adoption of TDNs enables improved biosensor response for as high as 98% as compared with single-stranded DNA probes. • DNA detection of AKT2 gene was achieved with a linear detection range from 1 pM to 1 μM and a detection limit of 2 fM. • The effect of TDNs' linker length on the biosensor performance was investigated. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is a cytosolic DNA sensor which is regarded as a potential target for antitumor immunotherapy. However, clinical trials of STING agonists display limited anti-tumor effects and dose-dependent side-effects like inflammatory damage and cell toxicity. Here, we showed that tetrahedral DNA nanostructures (TDNs) actively enter macrophages to promote STING activation and M1 polarization in a size-dependent manner, and synergized with Mn 2+ to enhance the expressions of IFN- β and iNOS, as well as the co-stimulatory molecules for antigen presentation. Moreover, to reduce the cytotoxicity of Mn 2+ , we constructed a TDN-MnO 2 complex and found that it displayed a much higher efficacy than TDN plus Mn 2+ to initiate macrophage activation and anti-tumor response both in vitro and in vivo . Together, our studies explored a novel immune activation effect of TDN in cancer therapy and its synergistic therapeutic outcomes with MnO 2 . These findings provide new therapeutic opportunities for cancer therapy., Competing Interests: The authors have no conflicts of interest to declare., (© 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

The limited reaction time and sample volume in the confined space of microfluidic devices give considerable importance to the development of more effective biosensing interfaces. Herein, the self-assembling of tetrahedral framework nucleic acids (FNAs) with controllable size on the interface of the microfluidic microchannels is studied. Compared with macroscopic turbulence control on traditional micro-structured microfluidic surface, the novel FNA-engineered microfluidic interface successfully constructs a 3D reaction space at nanoscale by raising DNA probes away from the surface. This FNA interface dramatically improves the reaction kinetics during molecular recognition due to extremely ordered orientation, configuration and density of DNA probes on the surface. Finally, the FNA-engineered interface is applied in a novel multi-functional microfluidic platform, towards a "one-stop" assay of Escherichia coli O157: H7 (E. coli O157: H7), integrating capture, release, enrichment, cell culture and antimicrobial susceptibility testing (AST). With the FNA-aptamer probe, we achieved an enhanced bacterial detecting efficiency (10 CFU/mL) plus excellent selectivity and precision. The appicability was strongly demonstrated when the biosensor was successfully applied in real samples, including the analysis of antibiotic susceptibility and minimum inhibitory concentration (MIC) of E. coli O157: H7 among different antibiotics. The application of FNA interface will open a wide avenue for the development of microfluidic biosensors for other pathogenic microorganisms or circulating tumor cells (CTC) simply by changing the aptamers. Image 1 • A tetrahedral framework nucleic acids-based aptasensing interface has been constructed in the microfluidic system. • The development of an integrated microfluidic platform for bacterial detection, cell culture, and AST activities. • A desirable detection sensitivity (as low as 10 CFU/mL) and specificity for E. coli O157: H7. • The platform was able to provide fast (within 5 h) and reliable analysis of AST. [ABSTRACT FROM AUTHOR]

• A tetrahedral DNA nanostruture is designed which act as a carrier for DNAzyme and an anti-endonuclease degradation scaffold for APE1 substrate. • A new fluorescent biosensors was developed to detect APE1 activity with high sensitivity and selectivity and screen APE1 effectors. • This method was successfully applied for monitoring intracellular APE1 activity changes caused by drugs. Human apurinic/apyrimidinic endonuclease 1 (APE1) is an emerging target for sensitizing tumor cells to radiation and chemotherapy due to its critical function in DNA repair and reduction-oxidation regulation. Thus, accurate detection and intracellular imaging of APE1 hold great promise for drug screening, cancer diagnosis and prognosis evaluation. Here, a DNAzyme modified tetrahedral DNA nanostructure (DZ-TDN) and molecular beacon (MB) incorporated biosensing system was developed to reliably monitor intracellular APE1 activity. DZ-TDN was self-assembled from four single-stranded DNAs, one of which contained an extrude DNAzyme sequence on the 5′-end. At normal status, DNAzyme activity was plugged by performing a duplex with a blocker strand containing an AP site. The digestion of APE1 on this site can restore DNAzyme activity, which consequently trigger MB cleavage recycle to amplify detection signal. The results indicated that the proposed fluorescence method showed a linear response ranged for APE1 from 0.01 to 1 U mL−1 with a limit of detection (LOD) of 0.01 U mL−1 (S/N = 3). Moreover, the strategy was successfully used for effectors screening and intracellular imaging of APE1 with high reliability. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction, which can be extremely difficult to treat, is the worst deadly disease around the world. Reperfusion is expedient to reverse myocardial ischemia. However, during reperfusion, reactive oxygen species (ROS) produced by myocardial ischemia-reperfusion injury (MIRI) and further cell apoptosis are the most serious challenges to cardiomyocytes. Therefore, searching for reagents that can simultaneously reduce oxidative damage and MIRI-induced apoptosis is the pivotal strategy to rescue injured cardiomyocytes. Nevertheless, current cardioprotective drugs have some shortcomings, such as cardiotoxicity, inadequate intravenous administration, or immature technology. Previous studies have shown that tetrahedral DNA nanostructures (TDNs) have biological safety with promising anti-inflammatory and antioxidative potential. However, the progress that TDNs have made in the biological behavior of cardiomyocytes has not been explored. In this experiment, a cellular model of MIRI was first established. Then, confirmed by a series of experiments, our study indicates that TDNs can significantly decrease oxidative damage and apoptosis by limiting the overexpression of ROS, along with effecting the expression of apoptosis-related proteins. In addition, Western blot analysis demonstrated that TDNs could activate the Akt/Nrf2 signaling pathway to improve the myocardial injury induced by MIRI. Above all, the antioxidant and antiapoptotic capacities of TDNs make them a potential therapeutic drug for MIRI. This study provides new ideas and directions for more homogeneous diseases induced by oxidative damage.

One of the biggest obstacles for the use of antisense oligonucleotides as antibacterial therapeutics is their limited uptake by bacterial cells without a suitable carrier, especially in multi-drug-resistant bacteria with a drug efflux mechanism. Existing vectors, such as cell-penetrating peptides, are inefficient and nontargeting, and accordingly are not ideal carriers. A noncytotoxic tetrahedral DNA nanostructure (TDN) with a controllable conformation has been developed as a delivery vehicle for antisense oligonucleotides. In this study, antisense peptide nucleic acids (asPNAs) targeting a specific gene ( ftsZ) were efficiently transported into methicillin-resistant Staphylococcus aureus cells by TDNs, and the expression of ftsZ was successfully inhibited in an asPNA-concentration-dependent manner. The delivery system specifically targeted the intended gene. This novel delivery system provides a better platform for future applications of antisense antibacterial therapeutics and provides a basis for the development of a new type of antibacterial drug for multi-drug-resistant bacterial infections.

Dental pulp stem cells (DPSCs) derived from the human dental pulp tissue have multiple differentiation capabilities, such as osteo/odontogenic differentiation. Therefore, DPSCs are deemed as ideal stem cell sources for tissue regeneration. As new nanomaterials based on DNA, tetrahedral DNA nanostructures (TDNs) have tremendous potential for biomedical applications. Here, the authors aimed to explore the part played by TDNs in proliferation and osteo/odontogenic differentiation of DPSCs, and attempted to investigate if these cellular responses could be driven by activating the canonical Notch signaling pathway. Upon exposure to TDNs, proliferation and osteo/odontogenic differentiation of DPSCs were dramatically enhanced, accompanied by up regulation of Notch signaling. In general, our study suggested that TDNs can significantly promote proliferation and osteo/odontogenic differentiation of DPSCs, and this remarkable discovery can be applied in tissue engineering and regenerative medicine to develop a significant and novel method for bone and dental tissue regeneration., (Copyright © 2018 Elsevier Inc. All rights reserved.)