Fabrication and evaluation of molecularly imprinted magnetic nanoparticles for selective recognition and magnetic separation of lysozyme in human urine.

In this study, lysozyme-based magnetic molecularly imprinted polymers (Lyz-MMIPs) for selective recognition and magnetic separation of lysozyme in human urine were prepared via surface imprinting technology. The morphology and structural properties of the resultant Lyz-MMIPs were characterized by using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), thermogravimetry (TG), X-ray diffraction (XRD) and a vibrating sample magnetometer (VSM). The results showed that the Lyz-MMIPs exhibited a uniform core-shell structure and favorable magnetic properties with a saturation magnetization of 14.8 emu g-1. To obtain the best selectivity and binding performance, the pH value of adsorption solution was investigated in detail. Under the optimized conditions, the Lyz-MMIPs possessed high binding and specific recognition capacity, as well as fast binding kinetics and phase separation rates. Moreover, the saturation binding capacity of Lyz-MMIPs reached 124.3 mg g-1, which was nearly 3.2 times that of lysozyme-based magnetic non-imprinted polymers (Lyz-MNIPs). In addition, the selectivity and reusability experiments showed that the Lyz-MMIPs displayed significant selectivity and favorable reusability. Furthermore, the Lyz-MMIPs were successfully applied for the determination and separation of lysozyme in human urine with satisfactory recovery rates. Above all, the synthetic process was quite simple and this strategy may provide a versatile approach for the fabrication of well-defined molecularly imprinted polymers on magnetic nanoparticles for the analysis of complicated matrixes.

Constructing Smart Protocells with Built-In DNA Computational Core to Eliminate Exogenous Challenge.

A DNA reaction network is like a biological algorithm that can respond to "molecular input signals", such as biological molecules, while the artificial cell is like a microrobot whose function is powered by the encapsulated DNA reaction network. In this work, we describe the feasibility of using a DNA reaction network as the computational core of a protocell, which will perform an artificial immune response in a concise way to eliminate a mimicked pathogenic challenge. Such a DNA reaction network (RN)-powered protocell can realize the connection of logical computation and biological recognition due to the natural programmability and biological properties of DNA. Thus, the biological input molecules can be easily involved in the molecular computation and the computation process can be spatially isolated and protected by artificial bilayer membrane. We believe the strategy proposed in the current paper, i.e., using DNA RN to power artificial cells, will lay the groundwork for understanding the basic design principles of DNA algorithm-based nanodevices which will, in turn, inspire the construction of artificial cells, or protocells, that will find a place in future biomedical research.

Aligner-mediated cleavage of nucleic acids and its application to isothermal exponential amplification.

We herein describe a simple and versatile approach to use conventional nicking endonuclease (NEase) for programmable sequence-specific cleavage of DNA, termed aligner-mediated cleavage (AMC), and its application to DNA isothermal exponential amplification (AMC-based strand displacement amplification, AMC-SDA). AMC uses a hairpin-shaped DNA aligner (DA) that contains a recognition site in its stem and two side arms complementary to target DNA. Thus, it enables the loading of an NEase on DA's stem, localization to a specific locus through hybridization of the side arms with target DNA, and cleavage thereof. By using just one NEase, it is easy to make a break at any specific locus and tune the cleavage site to the single-nucleotide scale. This capability also endows the proposed AMC-SDA with excellent universality, since the cleavage of target DNA, followed by a polymerase-catalyzed extension along a particular primer as a key step for initiating SDA, no longer relies on any special sequence. Moreover, this manner of initiation facilitates the adoption of 3'-terminated primers, thus making AMC-SDA highly sensitive and highly specific, as well as simple primer design.

Enhanced Targeted Gene Transduction: AAV2 Vectors Conjugated to Multiple Aptamers via Reducible Disulfide Linkages.

Enhanced targeted gene transduction by AAV2 vectors is achieved by linking the vector to multiple sgc8 aptamers, which are selective for cell membrane protein PTK7. Aptamer molecules are conjugated to multiple sites on a DNA dendrimer (G-sgc8), which is then linked to AAV2 via a dithiobis(succinimidyl propionate) cross-linker containing a disulfide group, which can facilitate the release of AAV2 vectors by reaction with the reduced form of intracellular glutathione. The G-sgc8-AAV2 vectors showed a 21-fold enhancement in binding affinity and an enhanced ability to protect sgc8 aptamers against nuclease degradation to cells expressing PTK7 compared to single aptamer-AAV2 conjugates. The transduction efficiency was tested by loading AAV2 with the gene for green fluorescent protein. Therefore, this modified recombinant vector is an attractive and promising tool for targeted biomedical applications.

Molecular Recognition-Based DNA Nanoassemblies on the Surfaces of Nanosized Exosomes.

Exosomes are membrane-enclosed extracellular vesicles derived from cells, carrying biomolecules that include proteins and nucleic acids for intercellular communication. Owning to their advantages of size, structure, stability, and biocompatibility, exosomes have been used widely as natural nanocarriers for intracellular delivery of theranostic agents. Meanwhile, surface modifications needed to endow exosomes with additional functionalities remain challenging by their small size and the complexity of their membrane surfaces. Current methods have used genetic engineering and chemical conjugation, but these strategies require complex manipulations and have only limited applications. Herein, we present an aptamer-based DNA nanoassemblies on exosome surfaces. This in situ assembly method is based on molecular recognition between DNA aptamers and their exosome surface markers, as well as DNA hybridization chain reaction initiated by an aptamer-chimeric trigger. It further demonstrated selective assembly on target cell-derived exosomes, but not exosomes derived from nontarget cells. The present work shows that DNA nanostructures can successfully be assembled on a nanosized organelle. This approach is useful for exosome modification and functionalization, which is expected to have broad biomedical and bioanalytical applications.

DNA probes for monitoring dynamic and transient molecular encounters on live cell membranes.

Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.

Generation of Biostable L-aptamers against Achiral Targets by Chiral Inversion of Existing D-aptamers.

In this paper, based on reciprocal chiral substrate specificity, taking achiral molecules, ethanolamine (EA) and malachite green (MG) as two model targets, biostable L- DNA aptamers and L-RNA aptamers were generated respectively by chiral inversion of existing D-aptamers. In the detection of EA with L-DNA aptamer-based sensors, the feasibility of our strategy was confirmed, while in the detection of MG with L-RNA aptamers, linear calibration curves were obtained in the range from 0.1 to 5µm with the detection limit of 0.065µm under optimized experimental conditions. The results demonstrated that the mirror-image L-aptamers have identical recognition capability as D-aptamers. Meanwhile, L-aptamers have superior biostability to resist nuclease digestion, protein binding interference and off-target effects, enabling their applications in complex practical samples, such as lake water and fish tissue extractions. Our work provides a simple, yet universal and efficient way to develop biostable aptamers.

Versatile surface engineering of porous nanomaterials with bioinspired polyphenol coatings for targeted and controlled drug delivery.

The development of biocompatible drug delivery systems with targeted recognition and controlled release has experienced a number of design challenges, including, for example, complicated preparation steps and premature drug release. Herein, we address these problems through an in situ self-polymerization method that synthesizes biodegradable polyphenol-coated porous nanomaterials for targeted and controlled drug delivery. As a proof of concept, we synthesized polyphenol-coated mesoporous silica nanoparticles, termed MSN@polyphenol. The polyphenol coatings not only improved colloidal stability and prevented premature drug leakage, but also provided a scaffold for immobilization of targeting moieties, such as aptamers. Both immobilization of targeting aptamers and synthesis of polyphenol coating are easily accomplished without the aid of any other organic reagents. Importantly, the polyphenol coating (EGCg) used in this study could be biodegraded by acidic pH and intracellular glutathione, resulting in the release of trapped anticancer drugs. Based on confocal fluorescence microscopy and cytotoxicity experiments, drug-loaded and polyphenol-coated MSNs were shown to possess highly efficient internalization and an apparent cytotoxic effect on target cancer, but not control, cells. Our results suggest that these highly biocompatible and biodegradable polyphenol-coated MSNs are promising vectors for controlled-release biomedical applications and cancer therapy.

DNA micelle flares: a study of the basic properties that contribute to enhanced stability and binding affinity in complex biological systems.

DMFs are spherical DNA-diacyllipid nanostructures formed by hydrophobic effects between lipid tails coupled to single-stranded DNAs. Such properties as high cellular permeability, low critical micelle concentration (CMC) and facile fabrication facilitate intracellular imaging and drug delivery. While the basic properties of NFs have been amply described and tested, few studies have characterized the fundamental properties of DMFs with particular respect to aggregation number, dissociation constant and biostability. Therefore, to further explore their conformational features and enhanced stability in complex biological systems, we herein report a series of characterization studies. Static light scattering (SLS) demonstrated that DMFs possess greater DNA loading capacity when compared to other DNA-based nanostructures. Upon binding to complementary DNA (cDNA), DMFs showed excellent dissociation constants (Kd) and increased melting temperatures, as well as constant CMC (10 nM) independent of DNA length. DMFs also present significantly enhanced stability in aqueous solution with nuclease and cell lysate. These properties make DMFs ideal for versatile applications in bioanalysis and theranostics studies.

Biostable L-DNAzyme for Sensing of Metal Ions in Biological Systems.

DNAzymes, an important type of metal ion-dependent functional nucleic acid, are widely applied in bioanalysis and biomedicine. However, the use of DNAzymes in practical applications has been impeded by the intrinsic drawbacks of natural nucleic acids, such as interferences from nuclease digestion and protein binding, as well as undesired intermolecular interactions with other nucleic acids. On the basis of reciprocal chiral substrate specificity, the enantiomer of D-DNAzyme, L-DNAzyme, could initiate catalytic cleavage activity with the same achiral metal ion as a cofactor. Meanwhile, by using the advantage of nonbiological L-DNAzyme, which is not subject to the interferences of biological matrixes, as recognition units, a facile and stable L-DNAzyme sensor was proposed for sensing metal ions in complex biological samples and live cells.

Designing a Biostable L-DNAzyme for Lead(II) Ion Detection in Practical Samples.

A promising biosensor for effectively lead (II) ion detection in practical applications was developed by constructing a Pb2+-specific L-DNAzyme, the enantiomer of the natural nucleic acid-constructed D-DNAzyme. This fluorescent sensor contains the L-enzyme strand with a quencher at the 3' end, and the L-substrate strand with a fluorophore at the 5' and a quencher at the 3' ends that formed a complex. In the presence of Pb2+, the L-substrate is cut into two fragments, leading to the recovery of fluorescence. The sensor shows high sensitivity and selectivity for Pb2+ detection with a linear response in the range of 5-100 nM and a detection limit of 3 nM in aqueous solution. Importantly, based on that L-DNAzyme consists of non-natural nucleic acids, which is insensitive to nuclease digestion, protein adsorption and D-DNA hybridization, our sensor shows specific response to Pb2+ in practical water and serum samples. Therefore, it is expected that our L-DNAzyme-based strategy may offer a new method for developing simple, rapid and sensitive sensors in complex systems.

Fabrication of Ultrathin Zn(OH)2 Nanosheets as Drug Carriers.

Ultrathin two-dimensional (2D) porous Zn(OH)2 nanosheets (PNs) have been fabricated by using one-dimensional Cu nanowires as backbones. PNs have thicknesses of about 3.8 nm and pore sizes of 4~10 nm. To form "smart" porous nanosheets, DNA aptamers were covalently conjugated on the surface of PNs. These ultrathin nanosheets show good biocompatibility, efficient cellular uptake, and promising pH-stimulated drug release.

Ionic Functionalization of Hydrophobic Colloidal Nanoparticles To Form Ionic Nanoparticles with Enzymelike Properties.

Inorganic colloidal nanoparticles (NPs) stabilized by a layer of hydrophobic surfactant on their surfaces have poor solubility in the aqueous phase, thus limiting their application as biosensors under physiological conditions. Here we report a simple model to ionize various types of hydrophobic colloidal NPs, including FePt, cubic Fe3O4, Pd, CdSe, and NaYF4 (Yb 30%, Er 2%, Nd 1%) NPs, to multicharged (positive and negative) NPs via ligand exchange. Surfaces of neutral hydrophobic NPs were converted to multicharged ions, thus making them soluble in water. Furthermore, peroxidase-like activity was observed for ionic FePt, Fe3O4, Pd, and CdSe NPs, of which FePt and CdSe catalyzed the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) to the blue-colored product in the absence of H2O2, while Pd and Fe3O4 catalyzed the oxidization of TMB in the presence of H2O2. With the benefit of the ionic functionalization protocols described herein, colloidal NPs should gain wider use as biomarkers, nanozymes, and biosensors.

Self-Assembled DNA Immunonanoflowers as Multivalent CpG Nanoagents.

Synthetic unmethylated cytosine-guanine (CpG) oligodeoxynucleotides are immunostimulatory motifs that have shown promise as vaccines or adjuvants for diseases such as cancers and infectious diseases. In the present work, novel immuno-nanoflowers (NFs), self-assembled from long DNA integrated with tandem CpG through rolling circle replication, were developed for efficient CpG delivery and protection from nuclease degradation. In a model of macrophage-like cells, the CpG NFs proved to be potent immunostimulators by triggering the proliferation of these immune cells, which, in turn, secreted immunostimulatory cytokines, including tumor necrosis factor α, interleukin-6, and interleukin-10. These results demonstrate the ability of CpG NFs to induce cancer cell apoptosis and necrosis.

Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers.

We describe a comprehensive protocol for the preparation of multifunctional DNA nanostructures termed nanoflowers (NFs), which are self-assembled from long DNA building blocks generated via rolling-circle replication (RCR) of a designed template. NF assembly is driven by liquid crystallization and dense packaging of building blocks, which eliminates the need for conventional Watson-Crick base pairing. As a result of dense DNA packaging, NFs are resistant to nuclease degradation, denaturation or dissociation at extremely low concentrations. By manually changing the template sequence, many different functional moieties including aptamers, bioimaging agents and drug-loading sites could be easily integrated into NF particles, making NFs ideal candidates for a variety of applications in biomedicine. In this protocol, the preparation of multifunctional DNA NFs with highly tunable sizes is described for applications in cell targeting, intracellular imaging and drug delivery. Preparation and characterization of functional DNA NFs takes ∼5 d; the following biomedical applications take ∼10 d.

A cascade reaction network mimicking the basic functional steps of adaptive immune response.

Biological systems use complex 'information-processing cores' composed of molecular networks to coordinate their external environment and internal states. An example of this is the acquired, or adaptive, immune system (AIS), which is composed of both humoral and cell-mediated components. Here we report the step-by-step construction of a prototype mimic of the AIS that we call an adaptive immune response simulator (AIRS). DNA and enzymes are used as simple artificial analogues of the components of the AIS to create a system that responds to specific molecular stimuli in vitro. We show that this network of reactions can function in a manner that is superficially similar to the most basic responses of the vertebrate AIS, including reaction sequences that mimic both humoral and cellular responses. As such, AIRS provides guidelines for the design and engineering of artificial reaction networks and molecular devices.

Electrochemical detection of type 2 diabetes mellitus-related SNP via DNA-mediated growth of silver nanoparticles on single walled carbon nanotubes.

Herein, we proposed a new electrochemical sensing strategy for T2DM-related SNP detection via DNA-mediated growth of AgNPs on a SWCNT-modified electrode. Coupled with RNase HII enzyme assisted amplification, this approach could realize T2DM-related SNP assay and be applied in crude extracts of carcinoma pancreatic ß-cell lines.

Two-Photon Sensing and Imaging of Endogenous Biological Cyanide in Plant Tissues Using Graphene Quantum Dot/Gold Nanoparticle Conjugate.

One main source of cyanide (CN(-)) exposure for mammals is through the plant consumption, and thus, sensitive and selective CN(-) detection in plants tissue is a significant and urgent work. Although various fluorescence probes have been reported for CN(-) in water and mammalian cells, the detection of endogenous biological CN(-) in plant tissue remains to be explored due to the high background signal and large thickness of plant tissue that hamper the effective application of traditional one-photo excitation. To address these issues, we developed a new two-photo excitation (TPE) nanosensor using graphene quantum dots (GQDs)/gold nanoparticle (AuNPs) conjugate for sensing and imaging endogenous biological CN(-). With the benefit of the high quenching efficiency of AuNPs and excellent two-photon properties of GQDs, our sensing system can achieve a low detection limit of 0.52 µM and deeper penetration depth (about 400 µm) without interference from background signals of a complex biological environment, thus realizing sensing and imaging of CN(-) in different types of plant tissues and even monitoring CN(-) removal in food processing. To the best of our knowledge, this is the first time for fluorescent sensing and imaging of CN(-) in plant tissues. Moreover, our design also provides a new model scheme for the development of two-photon fluorescent nanomaterial, which is expected to hold great potential for food processing and safety testing.

DNA Aptamer Based Nanodrugs: Molecular Engineering for Efficiency.

In the past two decades, the study of cancer therapy has gradually advanced to the "nano" era. Numerous novel nanomaterials armed with unique physical properties have been introduced into biomedical research. At the same time, functional nucleic acid molecules, especially aptamers, have aroused broad attention from the biomedical community. Benefiting from the advancement of molecular engineering strategies, it is now feasible to combine the cancer-specific recognition capability of aptamers with various other special functions of nanomaterials to develop cancer-specific drugs at the nanoscale. Nanodrugs are now offering an unprecedented opportunity to achieve the goal of efficient targeted delivery as well as controlled release. This review highlights some achievements made in multiple aptamer-based nanodrug systems that have emerged in recent years, including studies in the infant stage of "proof-of-concept".

DLISA: A DNAzyme-Based ELISA for Protein Enzyme-Free Immunoassay of Multiple Analytes.

A DNAzyme-based ELISA, termed DLISA, was developed as a novel protein enzyme-free, triply amplified platform, combining a catalytic and molecular beacon (CAMB) system with a cation exchange reaction for ultrasensitive multiplex fluorescent immunosorbent assay. Classical ELISA, which employs protein enzymes as biocatalysts to afford amplified signals, suffers from poor stability caused by the irreversible denaturation of these enzymes under harsh conditions, such as heat and acidity. Compared with proteins, nucleic acids are more stable and adaptable, and they can be easily produced using a commercial DNA synthesizer. Moreover, the catalytic and cleavage activities of DNAzyme can be achieved in solution; thus, no enzyme immobilization is needed for detection. Taken together, these attributes suggest that a DNAzyme-based ELISA detection approach will be more robust than current ELISA assays. Importantly, the proposed triply amplified DLISA immunoassay method shows ultrasensitive detection of such targets as human IgG with a detection limit of 2 fg/mL (3 × 10(-17) M), which is well within the range of many important disease biomarkers. DLISA can also be used to construct a sensing array for simultaneous multiplexed detection. With these merits, this high-throughput, stable, simple, sensitive, and low-cost multiplex fluorescence immunoassay shows promise for applications in clinical diagnosis.