DNA Nanospheres Research Articles

Highly sensitive and simultaneous detection of microRNAs in serum using stir-bar assisted magnetic DNA nanospheres-encoded probes

There are critical interests in the detection of microRNA (miRNA) because it can be a blood-borne biomarker, but analytical strategies are still limited by its small size, high sequence homology among family members and low abundance. In this work, three-dimensional magnetic DNA nanospheres were synthesized and immobilized on a gold stir-bar as encoded probes for miRNA capture and signal amplification. Electrochemical tags-labeled DNAs were immobilized on gold coated magnetic nanospheres via a hyperbranched hybridization chain reaction (HHCR). Subsequently, the magnetic DNA nanospheres were immobilized on the gold stir-bar as encoded probes. Target miRNAs were captured on the surface of the stir-bar by replacing the magnetic DNA nanospheres-encoded probes, and the probes were magnetically enriched for highly sensitive and selective electrochemical detection. The gold stir-bar assisted magnetic DNA nanospheres-encoded probes possess dual functions: They are as a nanocarrier to increase the loading amounts of HHCR products, and they are also a platform for efficient electrochemical signal amplification via magnetic enrichment. The method was successfully applied for the detection of miRNA21 and miRNA155 in a wide linear range of 5 fM to 2 nM, and with detection limits of 1.5 fM and 1.8 fM, respectively. The preliminary application of the method suggests that it has great potential in the detection of miRNAs in serum samples.

Target-Triggered Polymerization of Branched DNA Enables Enzyme-free and Fast Discrimination of Single-Base Changes.

SummarySingle-base changes lead to important biological and biomedical implications; however, the discrimination of single-base changes from normal DNA always remains a grand challenge. Herein we developed a DNA recognition and amplification system based on artificial branched DNA, namely, target-triggered polymerization (TTP), to realize enzyme-free and fast discrimination of single-base changes. Branched DNA as monomers rapidly polymerized into DNA nanospheres only with the trigger of specific DNA. Our TTP system worked reliably over a wide range of conditions. Remarkably, our TTP system was capable of discriminating base-changing DNA from normal DNA, including distinguishing 1–4 nucleotide changes and positions of single base, which was attributed to the significant amplification of small differences in hybridization thermodynamics and kinetics. We further proposed a theoretical method for calculating the hybridization probability of nucleic acids, which performed highly consistent with experimental results.

3D DNA nanosphere-based photoelectrochemical biosensor combined with multiple enzyme-free amplification for ultrasensitive detection of cancer biomarkers

In this work, a new 3D DNA nanosphere was ingeniously designed and fabricated, which was used to combine with multiple enzyme-free amplification strategy to develop a photoelectrochemical (PEC) biosensing platform for ultrasensitive detection of carcinoembryonic antigen (CEA). The 3D DNA nanostructure was self-assembled by base complementary pairing in a few minutes and rolling circle amplification (RCA) reaction. The intense photocurrent derived from Au NPs/ZnSe QDs can be effectively decreased by 3D DNA nanospheres assembled on the electrode, making photoelectric signal present “off” state. The specific binding of target CEA with its hairpin (HP1) aptamer opens HP1 structure, which initiated multiple enzyme-free strand displacement amplification (SDA) reaction and generated a large number of single strands DNA S1. Then S1 competitively binds to capture DNA on the electrode to release 3D DNA nanospheres, thus the photocurrent signal became “on” state for achieving amplified assay of target CEA. The proposed PEC biosensor exhibits excellent performance with a wide linear range of 1.0 fg/mL to 10 ng/mL and a low detection limit of 0.12 fg/mL for CEA, which was successfully applied for the assay of real serum samples with good precision. The reported strategy opens a new simple way for PEC biosensor using DNA nanostructure, showing huge potential in clinical application research.

Evaluation of chitosan/eudragit hybrid coating over cubic DNA nanospheres with superior stability and therapeutic outcomes

Diabetes T2 is a quite devastating metabolic disorder recently affecting both young as well as old patients who have a failure in maintaining their glycemic-levels postprandial. The sulphonylurea class of drugs further increase the beta-cells exhaustion leading to decreased ability of beta-cells to release insulin in-turn causing feedback activation of alpha-cells to release glucagon. These complications arisen in the T2 diabetics further complicate the management of the disease in an irreversible way. However DPP-4 inhibitors such as Vildagliptin (VL) not only causes incretin-mediated (GLP1 & GIP), glucose-dependent beta-cells activation postprandial, with decreased evidence of beta-cells exhaustion but also suppress glucagon secretion that further decrease the load on beta-cells to maintain glycemic levels. To effectively inhibit the di-peptidyl-peptidase-4 (DPP-4) enzyme and elevate the postprandial incretin-levels targeted sustained release vildagliptin nano-formulation was required which may reach the large intestine as well as to blood more effectively with increased potential of DPP-4 inhibition round the clock with improved glycemic levels. For this purpose, gastro-resistant chitosan/eudragit (cationic polymers) hybrid coating was applied around the VL loaded DNA cubes (negatively charged) through the electrostatic interactions to make Chit/Eud-DNA-VL nanospheres. Hybrid coating technique provided better size uniformity (35–120 nm diameter), stability and attainment of firm circular surface topology with Entrapment efficiency up to 92% and extended drug release up to 18 ± 2 h, as compared to our previously reported methods (40–2000 nm in diameter, Encapsulation efficiency 75–85% and extended drug release up to 12 ± 5 h). In vivo experiments also revealed the improved GLP-1 levels in the blood and glycemic control. So hybrid nanoparticles showed better gastric-resistance and prolonged VL release at the target site in our in vivo experiments with no damage to the pancreatic cells confirmed through post-treatment histological examination.

Chitosan-coated rectangular DNA nanospheres for better outcomes of anti-diabetic drug

Type 2 diabetes is a metabolic disorder in which the patient fails to control the glycemic level due to compromised functioning of pancreatic α and β cells. The control of these cells is regulated by the extent of incretin peptides (GLP1 and GIP). Performance of these peptides is affected due to the increased degradation by di-peptidyl-peptidase-4 (DPP-4) enzyme in diabetic patients. Vildagliptin (VL) is one of the potential DPP-4 inhibitors and helps in controlling the glycemic level, but to deliver the VL near its site of action (intestine and blood) in a sustained manner is important. For this purpose, we used chitosan (Chit) (cationic polymer) to coat the VL-loaded DNA rectangles (negatively charged) via electrostatic attractions to make Chit-DNA-VL nanospheres. According to our results, nanospheres formulated by this novel approach were uniformed, spherical, and stable with better size control (from 40 to 400 nm in diameter). Entrapment efficiency for VL drug was up to 85%. The release of VL was extended up to 12 ± 5 h. From observed incretin and glycemic level, we concluded that the nanospheres efficiently bypassed the gastric acidity improving glycemic control in Db-Db/mouse model. Histological experiments revealed Chit-DNA-VL nanospheres did not cause damage to pancreas associated with the sustained and prolonged release of VL.

Fluorescent hybrid nanospheres induced by single-stranded DNA and magnetic carbon quantum dots

Assembled DNA nanospheres were preparedviaself-assembly with magnetic CQDGd as the building blocks and negatively charged ssDNA as the assembly units.

Engineering Multifunctional DNA Hybrid Nanospheres through Coordination‐Driven Self‐Assembly

AbstractDeveloping simple and general approaches for the synthesis of nanometer‐sized DNA materials with specific morphologies and functionalities is important for various applications. Herein, a novel approach for the synthesis of a new set of DNA‐based nanoarchitectures through coordination‐driven self‐assembly of FeII ions and DNA molecules is reported. By fine‐tuning the assembly, Fe–DNA nanospheres of precise sizes and controlled compositions can be produced. The hybrid nanoparticles can be tailored for delivery of functional DNA to cells in vitro and in vivo with enhanced biological function. This highlights the potential of metal ion coordination as a tool for directing the assembly of DNA architectures, which conceptualizes a new pathway to expand the repertoire of DNA‐based nanomaterials. This methodology will advance both the fields of DNA nanobiotechnology and metal–ligand coordination chemistry.

Novel Sensitive Fluorometric Determination of Exonuclease I Using Polydopamine Nanospheres

ABSTRACTA novel fluorescence sensing platform based on polydopamine nanospheres and 6′-carboxyfluorescein labeled single-stranded DNA has been developed for monitoring the concentration of exonuclease I. Due to the interaction between single-stranded DNA and polydopamine nanospheres, the single-stranded DNA may be adsorbed on the surface of polydopamine nanospheres. The fluorescence of 6′-carboxyfluorescein was subsequently quenched by the polydopamine nanospheres through energy transfer or electron transfer. However, the 6′-carboxyfluorescein-labeled single-stranded DNA was specifically degraded by exonuclease I, producing mono or oligonucleotide fragments, which were not adsorbed by the polydopamine nanospheres, and thus the fluorescence signal was retained. The retained fluorescence of the sensing platform was found to be linear with the concentration of exonuclease I in the range of 0.15–10 U mL−1 with a detection limit of 0.05 U mL−1. In addition, the sensing platform was highly selective toward exonuclease I. Benefiting from the high efficiency and the simple design process, satisfactory performance has been successfully demonstrated for the determination of exonuclease I in complex samples.

Aptamer-functionalized DNA nanosphere as a stimuli-responsive nanocarrier

Smart nanocarriers that can respond to specific signals have recently gained attention in clinical research. Through the scaffolded DNA origami technique, we developed an aptamer-functionalized DNA nanosphere that could change its conformation upon binding to cancer-specific Mucin 1 protein (MUC1). A MUC1 aptamer was added into the lock-and-key system of the DNA nanosphere, and specific binding of the aptamer and MUC1 triggered the opening of the structure. A decrease in FRET signals demonstrated that the DNA nanosphere underwent structural change to the open conformation in response to the MUC1-containing cell lysate. Gold nanoparticles were also successfully encapsulated inside the cavity, implicating its loading ability. Our data confirmed that the aptamer-modified DNA nanosphere has proved to be a responsive nanocarrier and may serve as a promising candidate for cancer theranostic purposes.

Vildagliptin loaded triangular DNA nanospheres coated with eudragit for oral delivery and better glycemic control in type 2 diabetes mellitus

Diabetes mellitus type 2 is a multidimensional disease associated with poor glycemic control through compromised sensitivity of pancreatic islet α and β cells against glucose and dwindled secretion of insulin which is linked with the quantity of incretin hormones that are abridged by dipeptidyl peptidase-4 (DPP-4) in diseased condition. Vildagliptin (VG) inhibits DPP-4 therefore regulates the incretins that conversely maintains glycemic control. The safe reach and absorption of VG from intestine was dubious. Therefore we used Electrostatic Attraction Method to develop drug loaded DNA nanotechnology triangles coated by Eudragit (Eud) to make stable nanospheres of Vildagliptin (VG). We further analyzed the formulated nanospheres by AFM, XRD, DSC, SEM, TGA, ATR-FTIR and native PAGE. Additionally the efficacy of formulated nanospheres for drug release and glycemic control was assessed in Db/Db mouse. Our results showed that formulated nanospheres are smooth, spherical, stable and uniform in size ranging from 500 to 2000 nm with drug entrapment efficiency up to 95 ± 2% and extended drug release up to 15 ± 2 h. FTIR and DSC results confirmed the absence of VG-DNA-Eud interaction and XRD studies revealed a change in the crystalline status of the VG in nanospheres. Ex-vivo studies indicate that Eud-DNA-VG nanospheres effectively bypasses the acidic pH of the stomach and enhances glycemic control in Db/Db mouse without any risk of pancreatitis or pancreatic cancer. To the best of our knowledge, this is the first study conclusively reporting that VG loaded DNA Nano-architects coated with Eudragit are stable, safe and may improve therapeutic outcomes after oral delivery.

Decomposable quantum-dots/DNA nanosphere for rapid and ultrasensitive detection of extracellular respiring bacteria

Extracellular respiring bacteria (ERB) are a group of bacteria capable of transferring electrons to extracellular acceptors and have important application in environmental remediation. In this study, a decomposable quantum-dots (QDs)/DNA nanosphere probe was developed for rapid and ultrasensitive detection of ERB. The QDs/DNA nanosphere was self-assembled from QDs-streptavidin conjugate (QDs-SA) and Y-shaped DNA nanostructure that is constructed based on toehold-mediated strand displacement. It can release numerous fluorescent QDs-SA in immunomagnetic separation (IMS)-based immunoassay via simple biotin displacement, which remarkably amplifies the signal of antigen-antibody recognizing event. This QDs/DNA-nanosphere-based IMS-fluorescent immunoassay is ultrasensitive for model ERB Shewanella oneidensis, showing a wide detection range between 1.0 cfu/mL and 1.0 × 108 cfu/mL with a low detection limit of 1.37 cfu/mL. Moreover, the proposed IMS-fluorescent immunoassay exhibits high specificity, acceptable reproducibility and stability. Furthermore, the proposed method shows acceptable recovery (92.4–101.4%) for detection of S. oneidensis spiked in river water samples. The proposed IMS-fluorescent immunoassay advances an intelligent strategy for rapid and ultrasensitive quantitation of low-abundance analyte and thus holds promising potential in food, medical and environmental applications.

Synthesis of a multi-functional DNA nanosphere barcode system for direct cell detection.

Nucleic acid-based technologies have been applied to numerous biomedical applications. As a novel material for target detection, DNA has been used to construct a barcode system with a range of structures. This paper reports multi-functionalized DNA nanospheres (DNANSs) by rolling circle amplification (RCA) with several functionalized nucleotides. DNANSs with a barcode system were designed to exhibit fluorescence for coding enhanced signals and contain biotin for more functionalities, including targeting through the biotin-streptavidin (biotin-STA) interaction. Functionalized deoxynucleotide triphosphates (dNTPs) were mixed in the RCA process and functional moieties can be expressed on the DNANSs. The anti-epidermal growth factor receptor antibodies (anti-EGFR Abs) can be conjugated on DNANSs for targeting cancer cells specifically. As a proof of concept, the potential of the multi-functional DNANS barcode was demonstrated by direct cell detection as a simple detection method. The DNANS barcode provides a new route for the simple and rapid selective recognition of cancer cells.

547 - DNA nanosphere as a drug delivery system for cancer cells

547 - DNA nanosphere as a drug delivery system for cancer cells

DNA Microspheres Coated with Bioavailable Polymer as an Efficient Gene Expression Agent in Yeasts

Gene delivery is one of the steps necessary for gene therapy and for genetic modification. However, delivering DNA into cells is challenging due to its negative charge that leads to repulsion by the negative cell membrane. In the current research, DNA spheres with a DNA encoding to a certain gene were coated with bioavailable polymers, polyethylene imine (PEI) and polycaprolactone (PCL), in a short, one-step sonochemical reaction. The polymers were used in order to neutralize the negative charge of the DNA. Our study shows that the DNA nanospheres not only managed to penetrate the cell without causing it any damage, but also expressed the desired gene inside it.

Observation of Single-Protein and DNA Macromolecule Collisions on Ultramicroelectrodes.

Single-molecule detection is the ultimate sensitivity in analytical chemistry and has been largely unavailable in electrochemical analysis. Here, we demonstrate the feasibility of detecting electrochemically inactive single biomacromolecules, such as enzymes, antibodies, and DNA, by blocking a solution redox reaction when molecules adsorb and block electrode sites. By oxidizing a large concentration of potassium ferrocyanide on an ultramicroelectrode (UME, radius ≤150 nm), time-resolved, discrete adsorption events of antibodies, enzymes, DNA, and polystyrene nanospheres can be differentiated from the background by their "footprint". Further, by assuming that the mass transport of proteins to the electrode surface is controlled mainly by diffusion, a size estimate using the Stokes-Einstein relationship shows good agreement of electrochemical data with known protein sizes.

Self-assembled multifunctional DNA nanospheres for biosensing and drug delivery into specific target cells

Self-assembly of three dimensional nucleic acid nanostructures is of great significance in nanotechnology, biosensing and biomedicine. Herein we present a novel class of multifunctional and programmable DNA nanostructures, termed nanospheres (NSs), with monodispersity, dense compaction and uniform size (∼ 200 nm) using only four DNAs based on not only Watson-Crick base pair hybridization between single-stranded DNA but also liquid crystallization and dense packing from periodic DNA duplexes. Due to the diversity of the internal structures, the present NSs can easily evolve into other kinds of DNA assemblies, such as DNA spherical structures with a larger size and a rough surface via rolling circle replication (RCR). Importantly, the functional arms incorporated in building units can be readily designed for biosensing and targeted cancer therapy with high payload capacity and excellent biocompatibility. Therefore, the proposed NSs could lead to novel routes for nucleic acid self-assembly, promising versatile applications in biosensing and biomedicine.

Wrapping/unwrapping transition of double-stranded DNA in DNA-nanosphere complexes induced by multivalent anions.

Wrapping and unwrapping behaviors of double-stranded DNA around a positively charged nanosphere in solution are studied by using the coarse-grained molecular dynamics (CGMD) simulation method. When monovalent, divalent and trivalent anions are added to the DNA-nanosphere complex solution, double-stranded DNA binds with a nanosphere owing to strong electrostatic attraction. However, when tetravalent anions are added to the DNA-nanosphere complex solution, local charge inversion is observed for a high anion concentration of tetravalent anions and the double-stranded DNA can be unwrapped from the nanosphere because of the local charge inversion near the nanosphere. Moreover, the helical structure of DNA is damaged when double-stranded DNA wraps around the nanosphere and the helical structure can be rebuilt when the double-stranded DNA unwraps from the nanosphere. This study can help us understand how to control the release of DNA in DNA-nanosphere complexes.

Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection.

We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rolling circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products μL(-1) as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.

DNA Nanospheres with Microfluidics: A Promising Platform for Cancer Diagnosis?

The mortality rate from cancer in the USA reached more than 570,000 in 2012, or equal to more than 1500 Americans each day [1]. More than 90% of cancer deaths result from metastasis [2], hence methods for early-stage cancer detection are vital for the reduction of mortality and they will have a significant medical and social impact. However, according to the Early Detection Research Network of the National Cancer Institute, ‘there are no validated molecular biomarker tests for the early detection of any cancer’ [3], even though a handful of biomarkers have been approved by the US FDA. It is known that, during metastasis, some cancer cells escape from the primary tumor through exfoliation, entering the bloodstream and becoming circulating tumor cells (CTCs) in the peripheral blood stream [2,4]. Some of these CTCs acquire the capability to colonize secondary sites, spreading tumors to distant organs [2,4]. Therefore, CTCs have the potential to serve as important biomarkers for early diagnosis of cancer metastasis [2,4]. An additional benefit is that CTC enumeration is less invasive than biopsy, while providing a quantifiable method for cancer diagnosis and prognosis. However, CTCs are extremely rare; the number of CTCs is in the order of one to 100 cells in 1 ml of blood [4]. For comparison, other cells in 1 ml of blood include approximately 5 × 10 erythrocytes, 7.8 × 10 leukocytes and approximately 3.5 × 10 platelets [5]. Therefore, detection of CTCs in peripheral blood is a needlein-a-haystack challenge. Currently, CellSearch (Veridex LLC, NJ, USA) is the only FDAapproved method, and it relies on antibodycoated magnetic particles for isolation of CTCs from blood [4]. One of its key drawbacks is low capture efficiency. Microfluidics could play an important role in enhancing the capture efficiency of CTCs.

Multivalent DNA nanospheres for enhanced capture of cancer cells in microfluidic devices.

Isolation of circulating tumor cells (CTCs) from peripheral blood or cancer cells from bone marrow has significant applications in cancer diagnosis, therapy monitoring, and drug development. CTCs are cancer cells shed from primary tumors; they circulate in the bloodstream, leading to metastasis. The extraordinary rarity of CTCs in the bloodstream makes their isolation a significant technological challenge. Herein, we report the development of a platform combining multivalent DNA aptamer nanospheres with microfluidic devices for efficient isolation of cancer cells from blood. Gold nanoparticles (AuNPs) were used as an efficient platform for assembling a number of aptamers for high-efficiency cell capture. Up to 95 aptamers were attached onto each AuNP, resulting in enhanced molecular recognition capability. An increase of 39-fold in binding affinity was confirmed by flow cytometry for AuNP-aptamer conjugates (AuNP-aptamer) when compared with aptamer alone. With a laminar flow flat channel microfluidic device, the capture efficiency of human acute leukemia cells from a cell mixture in buffer increased from 49% using aptamer alone to 92% using AuNP-aptamer. We also employed AuNP-aptamer in a microfluidic device with herringbone mixing microstructures for isolation of leukemia cells in whole blood. The cell capture efficiency was also significantly increased with the AuNP-aptamer over aptamer alone, especially at high flow rates. Our results show that the platform combining DNA nanostructures with microfluidics has a great potential for sensitive isolation of CTCs and is promising for cancer diagnosis and prognosis.