Tetrahedral DNA Nanostructures Research Articles

An impedance labeling free electrochemical aptamer sensor based on tetrahedral DNA nanostructures for doxorubicin determination.

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 100.0nM, 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 3.0nM. 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.

DNA framework-controlled Poly(MOFs) as a visual platform for diagnosis of HER2-positive breast cancer

The controllable assembly of metal-organic frameworks (MOFs) nanomaterials is an effective means to enhance their application capabilities. In this paper, a novel method is reported to control MOFs assembly by using a DNA framework material, a tetrahedral DNA nanostructure (TDN) labeled with phosphate groups at the vertex. In particular, various well-defined superstructures can be tailored by regulating the number of phosphate groups that coordinate with catalytic Zr-MOFs to create polymeric MOFs superstructures (Poly(MOFs)), exhibiting excellent catalytic activities and molecular recognition ability as well as greatly amplified signal readout. Furthermore, based on the DNA framework-controlled Poly(MOFs), a colorimetric biosensor with excellent analytical performance is fabricated for improving the diagnosis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. It is demonstrated that the quantification of HER2 protein can be achieved at molecular level with the limit of detection (LOD) of 12 pg L-1 and cellular level with the LOD of 10 cells. More importantly, the detection of HER2 in the serum of breast cancer patients have been achieved, thus HER2-positive and triple-negative breast cancer patients can be well distinguished. Considering the substitutability of the target, this study shows great potential in biomedical research as well as clinical applications.

A universal electrochemical biosensor based on CRISPR/Cas12a and a DNA tetrahedron for ultrasensitive nucleic acid detection.

Herein, a universal nucleic acid analysis platform was constructed for sensitive and accurate detection of miRNA-155 and ctDNA using isothermal amplification-assisted CRISPR/Cas12a and a tetrahedral DNA nanostructure (TDN) supported sensing interface. Under the optimal experimental conditions, the prepared sensor achieved specific detection of miRNA-155 and ctDNA at as low as aM levels in 2.6 h. Furthermore, the platform was also successfully applied to human serum sample recovery experiments and cancer cell lysates, demonstrating outstanding reliability and accuracy. We firmly believe that this work provides a universal, sensitive, and practical tool for early clinical diagnosis.

Revolutionizing cancer therapy using tetrahedral DNA nanostructures as intelligent drug delivery systems

DNA nanostructures have surfaced as intriguing entities with vast potential in biomedicine, notably in the drug delivery area.

Evaluating early apoptosis-related cellular MiRNAs with an ultrasensitive electrochemiluminescence nanoplatform.

It is known that the abnormal expression of specific cellular miRNAs is closely related to cell apoptosis, and so monitoring the level change of these miRNAs can in principle be used to evaluate the process of apoptosis stimulated by drugs. Towards this goal, here we construct an ultrasensitive electrochemiluminescence (ECL) nanoplatform via the target miRNA-triggered immobilization of spherical nucleic acid enzymes (SNAzymes) onto tetrahedral DNA nanostructures on the electrode surface, which catalyzes the luminol-H2O2 reaction to output an ECL signal. This enables the sensitive and specific detection of two apoptosis-related miRNAs, miR-21 and miR-133a, with a detection limit of 33 aM. Furthermore, we employed the developed ECL nanoplatform to monitor the levels of these two miRNAs inside cancer cells stimulated by DOX, showing that the level of miR-21 decreases, while that of miR-133a increases in the early apoptotic cells. This difference highlights the distinct roles of the two target miRNAs, where miR-21 promotes the early apoptosis of cancer cells, whereas miR-133a suppresses it, providing new insight into cell physiological processes.

All-in-one fabrication of a ratiometric electrochemical aptasensor with tetrahedral DNA nanostructure for fumonisin B1 detection.

Here, we develop an all-in-one strategy for efficient assembly of an electrochemical aptasensor. A multifunctional structure based on a tetrahedral DNA nanostructure (TDN) was synthesized via a one-step annealing process, providing DNA fixation, target recognition, signal amplification and space regulation. Based on the integration of this multifunctional structure, the sensing interface was assembled in one step. A ratiometric aptasensor was constructed by anchoring methylene blue (MB) to the TDN and ferrocene (Fc) on the cDNA. Using the ratio of the currents obtained from Fc and MB as a measure, the developed aptasensor shows excellent analytical performance for fumonisin B1 detection. This strategy is universal and could simplify the fabrication of aptasensors.

Aptamer-Modified Tetrahedral DNA Nanostructures as Drug Delivery System for Cancer Targeted Therapy.

Improving the selective delivery and uptake efficiency of chemotherapeutic drugs remains a challenge for cancer-targeted therapy. In this work, a DNA tetrahedron is constructed as a targeted drug delivery system for efficient delivery of doxorubicin (Dox) into cancer cells. The DNA tetrahedron is composed of a tetrahedral DNA nanostructure (TDN) with two strands of AS1411 aptamer as recognition elements which can target the nucleolin protein on the cell membrane of cancer cells. The prepared DNA tetrahedron has a high drug-loading capacity and demonstrates pH-responsive Dox release properties. This enables efficient delivery of Dox into targeted cancer cells while reducing side effects on nontarget cells. The proposed drug delivery system exhibits significant therapeutic efficacy in vitro compared to free Dox. Accordingly, this work provides a good paradigm for developing a targeted drug delivery system for cancer therapy based on DNA tetrahedrons.

A “signal off-on” fluorescence bioassay based on 2D-MoS2-tetrahedral DNA bioconjugate for rapid virus detection

In this work we present the preparation of a 2D molybdenum disulphide nanosheets (2D-MoS2) and tetrahedral DNA nanostructures (TDNs) bioconjugate, and its application to the development of a bioassay for rapid and easy virus detection. The bioconjugate has been prepared by using TDNs carrying the capture probe labelled with 6-carboxyfluoresceine (6-FAM). As case of study to assess the utility of the assay developed, we have chosen the SARS-CoV-2 virus. Hence, as probe we have used a DNA sequence complementary to a region of the SARS-CoV-2 ORF1ab gene (TDN-ORF-FAM). This 6-FAM labelled capture probe is located on the top vertex of the tetrahedral DNA nanostructure, the three left vertices of TDNs have a thiol group. These TDNs are bounded to 2D-MoS2 surface through the three thiol groups, allowing the capture probe to be oriented to favour the biorecognition reaction with the analyte. This biorecognition resulting platform has finally been challenged to the detection of the SARS-CoV-2 ORF1ab gene sequence as the target model by measuring fluorescence before and after the hybridization event with a detection limit of 19.7fM. Furthermore, due to high sensitivity of the proposed methodology, it has been applied to directly detect the virus in nasopharyngeal samples of infected patients without the need of any amplification step. The developed bioassay has a wide range of applicability since it can be applied to the detection of any pathogen by changing the probe corresponding to the target sequence. Thus, a novel, hands-on strategy for rapid pathogen detection has proposed and has a high potential application value in the early diagnosis of infections causes by virus or bacteria.

Topology- and size-dependent binding of DNA nanostructures to the DNase I

The susceptibility of DNA nanomaterials to enzymatic degradation in biological environments is a significant obstacle limiting their broad applications in biomedicine. While DNA nanostructures exhibit some resistance to nuclease degradation, the underlying mechanism of this resistance remains elusive. In this study, the interaction of tetrahedral DNA nanostructures (TDNs) and double-stranded DNA (dsDNA) with DNase I is investigated using all-atom molecular dynamics simulations. Our results indicate that DNase I can effectively bind to all dsDNA molecules, and certain key residues strongly interact with the nucleic bases of DNA. However, the binding of DNase I to TDNs exhibits a non-monotonic behavior based on size; TDN15 and TDN26 interact weakly with DNase I (∼ − 75 kcal/mol), whereas TDN21 forms a strong binding with DNase I (∼ − 110 kcal/mol). Furthermore, the topological properties of the DNA nanostructures are analyzed, and an under-twisting (∼32°) of the DNA helix is observed in TDN15 and TDN26. Importantly, this under-twisting results in an increased width of the minor groove in TDN15 and TDN26, which primarily explains their reduced binding affinity to DNase I comparing to the dsDNA. Overall, this study demonstrated a novel mechanism for local structural control of DNA at the nanoscale by adjusting the twisting induced by length.

Aptamer-modified tetrahedral DNA nanostructure-immobilized liposome for specific gene delivery and potential cancer theragnostic.

Targeted delivery of therapeutic agents to cancer cells is crucial for effective cancer treatment without adverse effects. In this study, we developed a novel delivery carrier, Aptamer-modified tetrahedral DNA nanostructure (TDN) immobilized Liposome (ApTL), for specific delivery to nucleolin-overexpressing cancer cells. We demonstrated that targeted ApTL was highly effective in delivering plasmid and mRNA to nucleolin-overexpressing cancer cells compared to non-targeted ApTL with a non-specific aptamer. ApTL, which is highly negative and nano-sized, specifically delivered nucleic acids to MDA-MB-231 and HeLa cancer cells, primarily via lipid-raft-mediated endocytosis. Furthermore, the co-delivery of mRNA and doxorubicin resulted in increased apoptosis and reduced cancer cell viability. Interestingly, co-delivery of mRNA and Dox did not show a significant difference in EGFP expression at 24h but dramatically increased EGFP expression at 48h, making ApTL/mEGFP/Dox a promising candidate for detecting live cancer cells after targeted cancer drug treatment. Our results suggest that ApTL can be a promising tool for the targeted delivery of therapeutic agents to nucleolin-overexpressing cancer cells, providing a new strategy for cancer theragnostic.

Tetrahedral DNA Nanostructure-Engineered Paper-Based Electrochemical Aptasensor for Fumonisin B1 Detection Coupled with Au@Pt Nanocrystals as an Amplification Label.

Fumonisin B1 (FB1), as one of the highest toxicity mycotoxins, poses a serious threat to animal and human health, even at low concentrations. It is significant and challenging to develop a sensitive and reliable analytical device. Herein, a paper-based electrochemical aptasensor was designed utilizing tetrahedral DNA nanostructures (TDNs) to controllably anchor an aptamer (Apt), improving the recognition efficiency of Apt to its target. First, gold nanoparticles (AuNPs)@MXenes were used as a sensing substrate with good conductivity and modified on the electrode for immobilization of complementary DNA-TDNs (cDNA-TDNs). In the absence of FB1, numerous Apt-Au@Pt nanocrystals (NCs) was hybridized with cDNA and assembled on the sensing interface, which accelerated the oxidation of TMB with H2O2 and produced a highly amplified differential pulse voltammetry (DPV) signal. When the target FB1 specifically bound to its Apt, the electrochemical signal was decreased by releasing the Apt-Au@Pt NCs from double-stranded DNA (dsDNA). On account of the strand displacement reaction by FB1 triggering, the aptasensor had a wider dynamic linear range (from 50 fg/mL to 100 ng/mL) with a lower limit of detection (21 fg/mL) under the optimized conditions. More impressively, the designed FB1 aptasensor exhibited satisfactory performance in corn and wheat samples. Therefore, the TDN-engineered sensing platform opens an effective approach for sensitive and accurate analysis of FB1, holding strong potential in food safety and public health.

Plasmonic nanocavity-modulated electrochemiluminescence sensor for gastric cancer exosomal miRNA detection

Plasmonic nanocavity possessing highly light field confinement and electromagnetic field enhancement can concentrate and enhance the luminescence signal. The plasmonic nanocavity has the great potential value in biosensing research and improve analytical sensitivity. In this work, we constructed a plasmonic nanocavity between circular Au nanoplate-film and spherical Au nanoparticle with tetrahedral DNA nanostructures. The nanocavity structure can regulate the local density of optical states and provide the field restriction to enhance the spontaneous ECL radiation of PEDOT-S dots. Additionally, Au nanoparticle acted as nanoantenna which localized and modulated ECL to directional emission. Because the plasmonic nanocavity effectively collected and redistributed ECL signal, the emission was enhanced by 5.9 times with polarized characteristics. The proposed plasmonic nanocavity-based ECL sensor was further used to detect exosomal miRNA-223-3p in ascites. The detection results indicated the novel sensing strategy can assist early diagnosis of peritoneal metastasis of gastric cancer.

Nitrogen-doped carbon dot and DNA tetrahedron nanostructure based electrochemiluminescence aptasensor for AFB1 detection

An electrochemiluminescence (ECL) aptasensor with aptamer as recognition element, N-doped carbon dots (N-CDs) as luminophore, and DNA tetrahedral nanostructure (DTN) as carrier to loading abundant N-CDs for signal amplification was developed for ultra-sensitive aflatoxin B1 (AFB1) detection. The complementary DNA (cDNA) was firstly immobilized on the Au electrode (AuE) surface to hybridize with the AFB1 aptamer. In the presence of AFB1, it would competitively with cDNA for binding the aptamer, causing the exposure of cDNA. DTN modified with AFB1 aptamer on top and N-CDs on the four vertices would combine with cDNA onto the AuE surface to generate ECL signals due to electron transfer between co-reactants (K2S2O8) and N-CDs, which was positively related to the concentration of AFB1. Due to the high specificity of aptamer, the outstanding ECL performance of N-CDs and the enhanced signal output of DTN/N-CDs probes, the as-constructed ECL aptasensor realized excellent sensitivity with a low detection limit of 77 pg/mL and a wide linear concentration of 1.0 × 10−1-1.0 × 104 ng/mL for AFB1, as well as fascinating accuracy with acceptable recoveries of 89.54–111.0% in the spiked Coix seed matrices. Successful application for AFB1 detection in actual samples confirmed the extensive practicability of the ECL aptasensor, providing a universal platform for more trace contaminants by altering the corresponding aptamers.

Free PCR virus detection via few-layer bismuthene and tetrahedral DNA nanostructured assemblies

In this work we describe a highly sensitive method based on a biocatalyzed electrochemiluminescence approach. The system combines, for the first time, the use of few-layer bismuthene (FLB) as a platform for the oriented immobilization of tetrahedral DNA nanostructures (TDNs) specifically designed and synthetized to detect a specific SARS-CoV-2 gene sequence. In one of its vertices, these TDNs contain a DNA capture probe of the open reading frame 1 ab (ORF1ab) of the virus, available for the biorecognition of the target DNA/RNA. At the other three vertices, there are thiol groups that enable the stable anchoring/binding to the FLB surface. This novel geometry/approach enables not only the binding of the TDNs to surfaces, but also the orientation of the capture probe in a direction normal to the bismuthine surface so that it is readily accessible for binding/recognition of the specific SARS-CoV-2 sequence. The analytical signal is based on the anodic electrochemiluminescence (ECL) intensity of luminol which, in turn, arises as a result of the reaction with H2O2, generated by the enzymatic reaction of glucose oxidation, catalyzed by the biocatalytic label avidin-glucose oxidase conjugate (Av-GOx), which acts as co-reactant in the electrochemiluminescent reaction. The method exhibits a limit of detection (LOD) of 4.31 aM and a wide linear range from 14.4 aM to 1.00 μM, and its applicability was confirmed by detecting SARS-CoV-2 in nasopharyngeal samples from COVID-19 patients without the need of any amplification process.

A two-step electrochemical biosensor based on Tetrazyme for the detection of fibrin.

In this study, an electrochemical biosensor was constructed for the detection of fibrin, specifically by a simple two-step approach, with a novel artificial enzyme (Tetrazyme) based on the DNA tetrahedral framework as signal probe. The multichannel screen-printed electrode with the activated surface cannot only remove some biological impurities, but also serve as a carrier to immobilize a large number of antigen proteins. The DNA tetrahedral nanostructure was employed to ensure the high sensitivity of the probe for biological analysis. The hemin was chimeric into the G-quadruplex to constitute the complex with peroxidase catalytic activity (hemin/G4-DNAzyme), subsequently, Tetrazyme was formed through combining of this complex and DNA tetrahedral nucleic acid framework. The artificial enzyme signal probe formed by the covalent combination of the homing peptide (Cys-Arg-Glu-Lys-Ala, CREKA), which is the aptamer of fibrin and the new artificial enzyme is fixed on the surface of the multichannel carbon electrode by CREKA-specific recognition, so as to realize the sensitive detection of fibrin. The feasibility of sensing platform was validated by cyclic voltammetry (CV) and amperometric i-t curve (IT) methods. Effects of Tetrazyme concentration, CREKA concentrations and hybridization time on the sensor were explored. Under the best optimal conditions of 0.6μmol/L Tetrazyme, 80μmol/L CREKA, and 2.5h reaction time, the immunosensor had two linear detection ranges, 10-40nmol/L, with linear regression equation Y = 0.01487X-0.011 (R2 = 0.992), and 50-100nmol/L, with linear regression equation Y = 0.00137X+0.6405 (R2 = 0.998), the detection limit was 9.4nmol/L, S/N≥3. The biosensor could provide a new method with great potential for the detection of fibrin with good selectivity, stability, and reproducibility.

Poly-adenine-mediated tetrahedral DNA nanostructure with multiple target-recognition sites for ultrasensitive and rapid electrochemical detection of Aflatoxin B1

Tetrahedral DNA nanostructures (TDNs) are widely used in the development of electrochemical biosensors due to their structural stability, programmability, and strong interfacial orderliness. However, the complex modifications on the electrode and the single vertex target recognition of the TDNs limit their applications in electrochemical biosensing. Herein, we developed a universal detection system based on a novel polyadenine-based tetrahedral DNA nanostructure (ATDN) using Aflatoxin B1 (AFB1) as the model target for analysis. In the absence of target AFB1, the signal probes (SP) modified with ferrocene would be anchored by five aptamers on ATDN. The target capture by aptamers led to a release of SP from the electrode surface, resulting in a significant reduction of the electrochemical signal. This new nanostructure was not only dispensed with multi-step electrode modifications and strong mechanical rigidity but also had five modification sites which enhanced the detection sensitivity for the target. As a result, this biosensor shows good analytical performance in the linear range of 1 fg mL−1 to 1 ng mL−1, exhibiting a low detection limit of 0.33 fg mL−1. Satisfactory accuracy has also been demonstrated through good recoveries (95.2%–98.9%). The proposed new tetrahedral DNA nanostructure can provide a more rapid and sensitive alternative to previous electrochemical sensors based on the conventional TDN. Since DNA sequences can be designed flexibly, the sensing platform in this strategy can be extended to detect various targets in different fields.

Noninvasive Framework Nucleic Acid Eye Drops for Retinal Administration.

Intravitreal injection is widely employed for the treatment of retinal diseases. However, it suffers from various drawbacks, including ocular trauma, risk of infection, and poor patient compliance due to frequent administrations. Due to the presence of barriers such as the cornea, it has been a challenge to develop efficient noninvasive ophthalmic eye drops that can reach the retina. Framework nucleic acids (FNAs), known for their excellent biocompatibility and precise, controllable shape and size, have been extensively utilized in drug delivery application. Here, we report the development of size- and shape-resolved fluorescent DNA frameworks for noninvasive retinal administration. Results show that tetrahedral DNA nanostructures (TDNs) with an edge length of 20 bp can reach the retina within 6 h with the highest efficiency. Moreover, this delivery method exhibits excellent biocompatibility. Our findings provide an approach for the development of localized treatment strategies for retinal diseases using FNA-based nanocarriers.

Programming DNA walker and DNA tetrahedral nanostructures mediated CRISPR/Cas12a electrochemiluminescence biosystem and its biosensing application

A CRISPR/Cas12a electrochemiluminescence (ECL) biosensor based on the multiple sites DNA walker with controlled swing arm position, integrating Tribulus terrestris-like AuCu nanoterrestris (AuCu NTs) and DNA tetrahedral nanostructures (DTNs) is constructed for ultrasensitive detection of miRNA-21. The AuCu NTs was served as a co-reaction accelerant to promote the reduction of S2O82- at the cathode, which can be rapidly converted with S2O83•- and significantly enhance the ECL signal from graphitic carbon nitride nanosheets. Meanwhile, by virtue of the excellent stability of DNA tetrahedral nanostructure, the binding efficiency between single strand DNA and luminescent materials was improved. Afterward, DNA walker signal amplifier converted and amplified the target miRNA-21 into transitional DNA under the activity of Nt.BsmAI nicking endonuclease, thereby activating trans-cleavage ability of CRISPR/Cas12a and achieving the efficient annihilation of ECL signals was employed. As a proof of concept, the as-constructed biosensor demonstrated well specificity and sensitivity towards miRNA-21 with the range of 1.0 fM to 10 nM and the detection limit was 0.339 fM (S/N = 3). Therefore, the proposed strategy expanded the scope of application of CRISPR/Cas12a and demonstrated potential of diagnostic analysis.

On-Site-Activated Transmembrane Logic DNA Nanodevice Enables Highly Specific Imaging of Cancer Cells by Targeting Tumor-Related Nucleolin and Intracellular MicroRNA.

The ability to specifically image cancer cells is essential for cancer diagnosis; however, this ability is limited by the false positive associated with single-biomarker sensors and off-site activation of "always active" nucleic acid probes. Herein, we propose an on-site, activatable, transmembrane logic DNA (TLD) nanodevice that enables dual-biomarker sensing of tumor-related nucleolin and intracellular microRNA for highly specific cancer cell imaging. The TLD nanodevice is constructed by assembling a tetrahedral DNA nanostructure containing a linker (L)-blocker (B)-DNAzyme (D)-substrate (S) unit. AS-apt, a DNA strand containing an elongated segment and the AS1411 aptamer, is pre-anchored to nucleolin protein, which is specifically expressed on the membrane of cancer cells. Initially, the TLD nanodevice is firmly sealed by the blocker containing an AS-apt recognition zone, which prevents off-site activation. When the nanodevice encounters a target cancer cell, AS-apt (input 1) binds to the blocker and unlocks the sensing ability of the nanodevice for miR-21 (input 2). The TLD nanodevice achieves dual-biomarker sensing from the cell membrane to the cytoplasm, thereby ensuring cancer cell-specific imaging. This TLD nanodevice represents a promising strategy for the highly reliable analysis of intracellular biomarkers and a promising platform for cancer diagnosis and related biomedical applications.

Tetrahedral DNA nanostructure enhanced toehold-mediated strand displacement for highly sensitive electrochemiluminescence assay of CA125

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)32+/metal–organic framework (AuNPs/Ru/ZIF-MOF) signal probe. AuNPs and Ru(bpy)32+ 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)32+, 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)32+ 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.