An Intelligent DNA Nanorobot for Autonomous Anticoagulation.

Artificial nanorobots that can recognize molecular triggers and respond with programable operations provide an inspiring proof-of-principle for personalized theragnostic applications. We have constructed an intelligent DNA nanorobot for autonomous blood anticoagulation in human plasma. The DNA nanorobot comprises a barrel-shaped DNA nanostructure as the framework and molecular reaction cascades embedded as the computing core. This nanorobot can intelligently sense the concentration of thrombin in the local environment and trigger an autonomous anticoagulation when excess thrombin is present. The triggering concentration of thrombin at which the nanorobot responds can be tuned arbitrarily to avoid possible side effects induced by excess thrombin. This makes the nanorobot useful for autonomous anticoagulation in various medical scenarios and inspires a more efficient and safer strategy for future personalized medicine.

Stable DNA Nanomachine Based on Duplex-Triplex Transition for Ratiometric Imaging Instantaneous pH Changes in Living Cells.

DNA nanomachines are becoming useful tools for molecular recognition, imaging, and diagnostics and have drawn gradual attention. Unfortunately, the present application of most DNA nanomachines is limited in vitro, so expanding their application in organism has become a primary focus. Hence, a novel DNA nanomachine named t-switch, based on the DNA duplex-triplex transition, is developed for monitoring the intracellular pH gradient. Our strategy is based on the DNA triplex structure containing C(+)-G-C triplets and pH-dependent Förster resonance energy transfer (FRET). Our results indicate that the t-switch is an efficient reporter of pH from pH 5.3 to 6.0 with a fast response of a few seconds. Also the uptake of the t-switch is speedy. In order to protect the t-switch from enzymatic degradation, PEI is used for modification of our DNA nanomachine. At the same time, the dynamic range could be extended to pH 4.6-7.8. The successful application of this pH-depended DNA nanomachine and motoring spatiotemporal pH changes associated with endocytosis is strong evidence of the possibility of self-assembly DNA nanomachine for imaging, targeted therapies, and controllable drug delivery.

Nucleic acid based logical systems.

Researchers increasingly visualize a significant role for artificial biochemical logical systems in biological engineering, much like digital logic circuits in electrical engineering. Those logical systems could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expression in vivo. Nucleic acids (NA), as carriers of genetic information with well-regulated and predictable structures, are promising materials for the design and engineering of biochemical circuits. A number of logical devices based on nucleic acids (NA) have been designed to handle various processes for technological or biotechnological purposes. This article focuses on the most recent and important developments in NA-based logical devices and their evolution from in vitro, through cellular, even towards in vivo biological applications.

Aptamer-incorporated hydrogels for visual detection, controlled drug release, and targeted cancer therapy.

Hydrogels are water-retainable materials, made from cross-linked polymers, that can be tailored to applications in bioanalysis and biomedicine. As technology advances, an increasing number of molecules have been used as the components of hydrogel systems. However, the shortcomings of these systems have prompted researchers to find new materials that can be incorporated into them. Among all of these emerging materials, aptamers have recently attracted substantial attention because of their unique properties, for example biocompatibility, selective binding, and molecular recognition, all of which make them promising candidates for target-responsive hydrogel engineering. In this work, we will review how aptamers have been incorporated into hydrogel systems to enable colorimetric detection, controlled drug release, and targeted cancer therapy.

Using photons to manipulate enzyme inhibition by an azobenzene-modified nucleic acid probe.

The ability to inhibit an enzyme in a specific tissue with high spatial resolution combined with a readily available antidote should find many biomedical applications. We have accomplished this by taking advantage of the cis-trans photoisomerization of azobenzene molecules. Specifically, we positioned azobenzene moieties within the DNA sequence complementary to a 15-base-long thrombin aptamer and then linked the azobenzene-modified cDNA to the aptamer by a polyethylene glycol (PEG) linker to make a unimolecular conjugate. During the photoisomerization of azobenzene by visible light, the inhibition of thrombin is disabled because the probe hybridizes with the cDNA in the trans-azobenzene conformation so that the aptamer cannot bind its target thrombin. However, when UV light is applied, melting of the hairpin structure (duplex) is induced via trans-to-cis conversion, thereby changing conformation of the aptamer and making the aptamer free to bind to and inhibit its target thrombin. By using standard clotting assays, we measured the IC(200) of various probe designs in both states and concluded the feasibility of using photon energy to temporally and spatially regulate these enzymatic reactions. Thus, we can report the development of DNA probes in the form of photon-controllable (thrombin) inhibitors, termed PCIs, and we expect that this approach will be highly beneficial in future biomedical and pharmaceutical applications.

Photoresponsive DNA-cross-linked hydrogels for controllable release and cancer therapy.

We have developed a photoresponsive DNA-cross-linked hydrogel that can be photoregulated by two wavelengths with a reversible sol-gel conversion. This photoinduced conversion can be further utilized for precisely controllable encapsulation and release of multiple loads. Specifically, photosensitive azobenzene moieties are incorporated into DNA strands as cross-linkers, such that their hybridization to complementary DNAs (cDNAs) responds differently to different wavelengths of light. On the basis of the rheology variation of hydrogels, it is possible to utilize this material for storing and releasing molecules and nanoparticles. To prove the concept, three different materials--fluorescein, horseradish peroxidase, and gold nanoparticles--were encapsulated inside the gel at 450 nm and then released by photons at 350 nm. Further experiments were carried out to deliver the chemotherapy drug doxorubicin in a similar manner in vitro. Our results show a net release rate of 65% within 10 min, and the released drug maintained its therapeutic effect. This hydrogel system provides a promising platform for drug delivery in targeted therapy and in biotechnological applications.

A surface energy transfer nanoruler for measuring binding site distances on live cell surfaces.

Measuring distances at molecular length scales in living systems is a significant challenge. Methods like Förster resonance energy transfer (FRET) have limitations due to short detection distances and strict orientations. Recently, surface energy transfer (SET) has been used in bulk solutions; however, it cannot be applied to living systems. Here, we have developed an SET nanoruler, using aptamer-gold nanoparticle conjugates with different diameters, to monitor the distance between binding sites of a receptor on living cells. The nanoruler can measure separation distances well beyond the detection limit of FRET. Thus, for the first time, we have developed an effective SET nanoruler for live cells with long distance, easy construction, fast detection, and low background. This is also the first time that the distance between the aptamer and antibody binding sites in the membrane protein PTK7 was measured accurately. The SET nanoruler represents the next leap forward to monitor structural components within living cell membranes.

DNA-based micelles: synthesis, micellar properties and size-dependent cell permeability.

Functional nanomaterials based on molecular self-assembly hold great promise for applications in biomedicine and biotechnology. However, their efficacy could be a problem and can be improved by precisely controlling the size, structure, and functions. This would require a molecular engineering design capable of producing monodispersed functional materials characterized by beneficial changes in size, shape, and chemical structure. To address this challenge, we have designed and constructed a series of amphiphilic oligonucleotide molecules. In aqueous solutions, the amphiphilic oligonucleotide molecules, consisting of a hydrophilic oligonucleotide covalently linked to hydrophobic diacyllipid tails, spontaneously self-assemble into monodispersed, three-dimensional micellar nanostructures with a lipid core and a DNA corona. These hierarchical architectures are results of intermolecular hydrophobic interactions. Experimental testing further showed that these types of micelles have excellent thermal stability and their size can be fine-tuned by changing the length of the DNA sequence. Moreover, in the micelle system, the molecular recognition properties of DNA are intact, thus, our DNA micelles can hybridize with complimentary sequences while retaining their structural integrity. Importantly, when interacting with cell membranes, the highly charged DNA micelles are able to disintegrate themselves and insert into the cell membrane, completing the process of internalization by endocytosis. Interestingly, the fluorescence was found accumulated in confined regions of cytosole. Finally, we show that the kinetics of this internalization process is size-dependent. Therefore, cell permeability, combined with small sizes and natural nontoxicity are all excellent features that make our DNA-micelles highly suitable for a variety of applications in nanobiotechnology, cell biology, and drug delivery systems.

Single-DNA molecule nanomotor regulated by photons.

We report the design of a single-molecule nanomotor driven by photons. The nanomotor is a DNA hairpin-structured molecule incorporated with azobenzene moieties to facilitate reversible photocontrollable switching. Upon repeated UV-vis irradiation, this nanomotor displayed 40-50% open-close conversion efficiency. This type of nanomotor displays well-regulated responses and can be operated under mild conditions with no output of waste. In contrast to multiple-component DNA nanomachines, the intramolecular interaction in this single-molecule system offers unique concentration-independent motor functionality. Moreover, the hairpin structure of the motor backbone can significantly improve the efficiency of light-to-movement energy conversion. These results suggest that azobenzene-incorporated, hairpin-structured single-molecule DNA nanomotors have promising potential for applications which require highly efficient light-driven molecular motors.

Understanding Protection Mechanisms of Graphene-Encapsulated Silicon Anodes with Operando Raman Spectroscopy.

Carbon-coated silicon micro- and nanostructures have been widely used as composite anodes for lithium-ion batteries combining the benefits of high theoretical capacity of Si and better conductivity of carbon. To optimize structures that allow the Si volume expansion without losing the electrical connection, a detailed carbon protection mechanism is desired. We fabricate a network of interconnected sandwich branches with a silicon thin film encapsulated between a porous 3-dimensional graphene foam and graphene drapes (so-called a graphene ensemble). This prototype binder-free anode, of great mechanical strength and composed of only silicon and few-layer graphene, provides distinct signals under operando Raman spectroscopy. During electrochemical cycles, the graphene G peak shows variation of peak position and intensity, while the 2D peak experiences a negligible shift from limited deformation. Silicon displays excellent structural reversibility under the sandwich protection, validating the functions of graphenic carbon coating. This specific graphene ensemble can also serve as an experimental scaffold for mechanical and chemical analysis of many active materials.

Dynamic colloidal nanoparticle assembly triggered by aptamer-receptor interactions on live cell membranes.

Cells use dynamic systems such as enzyme cascades and signaling networks to control cellular functions. Synthetic dynamic systems that can be target-responsive have great potential to be applied for biomedical applications but the operation of such dynamic systems in complex cellular environments remains challenging. Here, we engineered an aptamer and DNA displacement reaction-based dynamic system that can transform its nanostructure in response to the epithelial cell adhesion molecule (EpCAM) on live cell membranes. The dynamic system consisted of a core nanoparticle and small satellite nanoparticles. With the modifications of different DNA hairpin strands and swing arm strands partially hybridized with an aptamer that specifically recognizes the EpCAM, the two separated particles can dynamically assemble into a core-satellite assembly by aptamer-receptor interactions on the cell membrane surface. The structural change of the system from separated particles to a core-satellite assembly generated plasmonic coupled hot spots for surface-enhanced Raman scattering (SERS) for sensitively capturing the dynamic structural change of the nanoassembly in the cellular environment. These concepts provide strategies for engineering dynamic nanotechnology systems for biological and biomedical applications in complex biological environments.

Engineering target-responsive hydrogels based on aptamer-target interactions.

In this communication, we report a simple, but highly adaptable, method of constructing selective target-responsive hydrogels using DNA aptamers. The simplicity of the design is accomplished by using linear polymer chains as the hydrogel backbone and a DNA aptamer as the cross-linker. In this design, competitive binding of target to the aptamer causes the decrease of cross-linking density and, hence, dissolution of the hydrogel. The adaptability of this strategy for therapeutic applications was demonstrated using two different types of targets, small molecules and proteins. Our results indicated that this molecular engineering provides a highly selective and controllable release system whereby efficient release of therapeutic agents can occur at specific environments in which the target biomarker is found.

Carbon nanotube-quenched fluorescent oligonucleotides: probes that fluoresce upon hybridization.

We report an effective, novel self-assembled single-wall carbon nanotube (SWNT) complex with an oligonucleotide and demonstrate its feasibility in recognizing and detecting specific DNA sequences in a single step in a homogeneous solution. The key component of this complex is the hairpin-structured fluorescent oligonucleotide that allows the SWNT to function as both a "nanoscaffold" for the oligonucleotide and a "nanoquencher" of the fluorophore. Given this functionality, this carbon nanotube complex represents a new class of universal fluorescence quenchers that are substantially different from organic quenchers and should therefore have many applications in molecular engineering and biosensor development. Competitive binding of a DNA target and SWNTs with the oligonucleotide results in fluorescence signal increments relative to the fluorescence without a target as well as in marked fluorescence quenching. In contrast to the common loop-and-stem configuration of molecular beacons (MBs), this novel fluorescent oligonucleotide needs only one labeled fluorophore, yet the emission can be measured with little or no background interference. This property greatly improves the signal-to-background ratio compared with those for conventional MBs, while the DNA-binding specificity is still maintained by the MB. To test the interaction mechanisms of the fluorescent oligonucleotide with SWNTs and target DNA, thermodynamic analysis and fluorescence anisotropy measurements, respectively, were applied. Our results show that MB/SWNT probes can be an excellent platform for nucleic acid studies and molecular sensing.

Noncovalent assembly of carbon nanotubes and single-stranded DNA: an effective sensing platform for probing biomolecular interactions.

In this paper, we report the assembly of single-walled carbon nanotubes (SWNTs) and single-stranded DNA to develop a new class of fluorescent biosensors which are able to probe and recognize biomolecular interactions in a homogeneous format. This novel sensing platform consists of a structure formed by the interaction of SWNTs and dye-labeled DNA oligonucleotides such that the proximity of the nanotube to the dye effectively quenches the fluorescence in the absence of a target. Conversely, and very importantly, the competitive binding of a target DNA or protein with SWNTs for the oligonucleotide results in the restoration of fluorescence signal in increments relative to the fluorescence without a target. This signaling mechanism makes it possible to detect the target by fluorescence spectroscopy. In the present study, the schemes for such fluorescence changes were examined by fluorescence anisotropy and fluorescence intensity measurements for DNA hybridization and aptamer-protein interaction studies.

Preparation of porous zinc ferrite/carbon as a magnetic-assisted dispersive miniaturized solid phase extraction sorbent and its application.

In this study, porous ZnFe2O4/carbon, derived from Zn-Fe zeolitic imidazolate framework (Zn-Fe-ZIF), was employed as a novel sorbent for magnetic-assisted dispersive miniaturized solid phase extraction (M-DµSPE). The Zn-Fe-ZIF derived magnetic porous ZnFe2O4/carbon was easily prepared using a one-pot solvothermal method, and its morphology, structure and magnetic characteristics were evaluated via scanning electron microscopy, powder X-ray diffraction, Raman spectroscopy and vibrating sample magnetometry. The extraction ability of ZnFe2O4/carbon is evaluated by different kinds of compounds including organochlorine pesticides, pyrethroid insecticides, aldehydes, nerolidol, benzoic acid and sorbic acid. A M-DµSPE method was developed for the analysis of organochlorine pesticides. Several parameters affecting the extraction efficiency were systematically investigated. The calibration curves ranged from 0.05 to 100 ng g-1 and the limits of detection were 0.005-0.3 ng g-1. The intra-day and inter-day relative standard deviations were lower than 2.3 and 5.2%. The recoveries of spiked organochlorine pesticides were in the range of 86.1-109.4%.

A cross-reactive sensor array for the fluorescence qualitative analysis of heavy metal ions.

A cross-reactive sensor array using mercaptopropionic acid modified cadmium telluride (CdTe), glutathione modified CdTe, poly(methacrylic acid) modified silver nanoclusters, bovine serum albumin modified gold nanoclusters, rhodamine derivative and calcein blue as fluorescent indicators has been designed for the detection of seven heavy metal ions (Ag(+), Hg(2+), Pb(2+), Cu(2+), Cr(3+), Mn(2+) and Cd(2+)). The discriminatory capacity of the sensor array to different heavy metal ions in different pH solutions has been tested and the results have been analyzed with linear discriminant analysis. Results showed that the sensor array could be used to qualitatively analyze the selected heavy metal ions. The array performance was also evaluated in the identification of known and unknown samples and the preliminary results suggested the promising practicability of the designed sensor assay.

Synergetic approach for simple and rapid conjugation of gold nanoparticles with oligonucleotides.

Attaching thiolated DNA on gold nanoparticles (AuNPs) has been extremely important in nanobiotechnology because DNA-AuNPs combine the programmability and molecular recognition properties of the biopolymers with the optical, thermal, and catalytic properties of the inorganic nanomaterials. However, current standard protocols to attach thiolated DNA on AuNPs involve time-consuming, tedious steps and do not perform well for large AuNPs, thereby greatly restricting applications of DNA-AuNPs. Here we demonstrate a rapid and facile strategy to attach thiolated DNA on AuNPs based on the excellent stabilization effect of mPEG-SH on AuNPs. AuNPs are first protected by mPEG-SH in the presence of Tween 20, which results in excellent stability of AuNPs in high ionic strength environments and extreme pHs. A high concentration of NaCl can be applied to the mixture of DNA and AuNP directly, allowing highly efficient DNA attachment to the AuNP surface by minimizing electrostatic repulsion. The entire DNA loading process can be completed in 1.5 h with only a few simple steps. DNA-loaded AuNPs are stable for more than 2 weeks at room temperature, and they can precisely hybridize with the complementary sequence, which was applied to prepare core-satellite nanostructures. Moreover, cytotoxicity assay confirmed that the DNA-AuNPs synthesized by this method exhibit lower cytotoxicity than those prepared by current standard methods. The proposed method provides a new way to stabilize AuNPs for rapid and facile loading thiolated DNA on AuNPs and will find wide applications in many areas requiring DNA-AuNPs, including diagnosis, therapy, and imaging.

Cancer cell sensing and therapy using affinity tag-conjugated gold nanorods.

Through the developments in controlling the shape of gold nanoparticles, synthesis of gold nanorods (AuNRs) can be considered as a milestone discovery in the area of nanomaterial-based cancer treatments. Besides having tuneable absorption maxima at near infrared (NIR) range, AuNRs have superior absorption cross section at NIR frequencies compared with other gold nanoparticles. When this unique optical property is combined with the specificity against cancer cells used by affinity tag conjugations, AuNRs become one of the most important nanoparticles used in both cancer cell sensing and in therapy. In this review, the impact of size and shape control of nanoparticles, especially AuNRs, on cancer cell treatments and a range of aptamer-conjugated AuNR applications in this regard are reviewed.