Fluorescence shadow imaging of Hypsibius exemplaris reveals morphological differences between sucrose- and CaCl2-induced osmobiotes.

Tardigrades are renowned for their ability to survive a wide array of environmental stressors. In particular, tardigrades can curl in on themselves while losing a significant proportion of their internal water content to form a structure referred to as a tun. In surviving varying conditions, tardigrades undergo distinct morphological transformations that could indicate different mechanisms of stress sensing and tolerance specific to the stress condition. Methods to effectively distinguish between morphological transformations, including between tuns induced by different stress conditions, are lacking. Herein, an approach for discriminating between tardigrade morphological states is developed and utilized to compare sucrose- and CaCl2-induced tuns, using the model species Hypsibius exemplaris. A novel approach of shadow imaging with confocal laser scanning microscopy enabled production of three-dimensional renderings of Hys. exemplaris in various physiological states resulting in volume measurements. Combining these measurements with qualitative morphological analysis using scanning electron microscopy revealed that sucrose- and CaCl2-induced tuns have distinct morphologies, including differences in the amount of water expelled during tun formation. Further, varying the concentration of the applied stressor did not affect the amount of water lost, pointing towards water expulsion by Hys. exemplaris being a controlled process that is adapted to the specific stressors.

In situ preparation of molybdenum-dioxide-incorporated carbonized silk fiber and its application in supercapacitors.

A previous study found that the capacitive behavior of nanoparticles fed to the silkworm can be delivered to carbonized silk fibers, which can be used to fabricate electrodes for the construction of flexible supercapacitors. However, the tendency of nanoparticles to aggregate decreases the quantity of nanoparticles that enter the silk and therefore reduces the capacitance performance of the prepared carbonized silk. Here, we sprayed ammonium molybdate tetrahydrate (AMT) on the surface of mulberry leaves used for feeding silkworms and investigated the effect of feeding AMT on the growth of silkworms and the properties of spun silk. The precursor incorporated into the silk was converted into scattered MoO2 NPs, which were embedded within the carbonized silk fiber (CSF) via carbothermal reduction. The specific capacitance of CSF obtained from silkworms fed with an aqueous solution of AMT-treated mulberry leaves reached up to 298 F/g at 0.2 g/A, which is much higher than that of the control group (102 F/g). Since AMT is highly water-soluble, and its concentration can be easily modulated, we believe that the proposed strategy is feasible for the large-scale fabrication of CSF with enhanced capacitive performance.

Point-of-care cancer diagnostic devices: From academic research to clinical translation.

Early and timely diagnosis of cancer plays a decisive role in appropriate treatment and improves clinical outcomes, improving public health. Significant advances in biosensor technologies are leading to the development of point-of-care (POC) diagnostics, making the testing process faster, easier, cost-effective, and suitable for on-site measurements. Moreover, the incorporation of various nanomaterials into the sensing platforms has yielded POC testing (POCT) platforms with enhanced sensitivity, cost-effectiveness and simplified detection schemes. POC cancer diagnostic devices provide promising platforms for cancer biomarker detection as compared to conventional in vitro diagnostics, which are time-consuming and require sophisticated instrumentation, centralized laboratories, and experienced operators. Current innovative approaches in POC technologies, including biosensors, smartphone interfaces, and lab-on-a-chip (LOC) devices are expected to quickly transform the healthcare landscape. However, only a few cancer POC devices (e.g. lateral flow platforms) have been translated from research laboratories to clinical care, likely due to challenges include sampling procedures, low levels of sensitivity and specificity in clinical samples, system integration and signal readout requirements. In this review, we emphasize recent advances in POC diagnostic devices for cancer biomarker detection and discuss the critical challenges which must be surmounted to facilitate their translation into clinical settings.

A review on nanomaterial-based field effect transistor technology for biomarker detection.

Field effect transistor (FET) based sensors have attractive features such as small size, ease of mass production, high versatility and comparably low costs. Over the last decade, many FET type biosensors based on various nanomaterials (e.g. silicon nanowires, graphene, and transition metal dichalcogenides) have been developed to detect various classes of biomolecular targets due to their integration into portable and rapid test systems, both for use in the clinical lab and in point-of-care testing. This review (with 197 refs.) starts with an introduction into the specific features of FET biosensor technology. This is followed by a description of the essentials of methods for immobilization of recognition elements. The next section discusses the progress that has been made in FET based biosensors using semiconducting nanostructures composed of silicon, graphene, metal oxides, and transition metal dichalcogenides. A further section is devoted to microfluidic systems combined with FET biosensors. We then emphasize the biosensing applications of these diagnostic devices for analysis of clinically relevant biomarkers, specifically to sensing of neurotransmitters, metabolites, nucleic acids, proteins, cancer and cardiac biomarkers. Two tables are presented which summarize advances in applications of 1D and 2D nanomaterial-based FETs for biomarker sensing. A concluding section summarizes the current status, addresses current challenges, and gives perspective trends for the field. Graphical abstract Field effect transistor devices based on the use of 1D and 2D semiconductor nanostructures (so called nano-FETs) are making use of materials including silicon nanowires, graphene, zinc oxide, indium oxide, titanium oxide, and molybdenum disulfide that are further modified with recognition elements for biosensing application.

Origami Arrays as Substrates for the Determination of Reaction Kinetics Using High-Speed Atomic Force Microscopy.

DNA nanostructures (DN) are powerful platforms for the programmable assembly of nanomaterials. As applications for DN both as a structural material and as a support for functional biomolecular sensing systems develop, methods enabling the determination of reaction kinetics in real time become increasingly important. In this report, we present a study of the kinetics of streptavidin binding onto biotinylated DN constructs enabled by these planar structures. High-speed AFM was employed at a 2.5 frame/s rate to evaluate the kinetics and indicates that the binding fully saturates in less than 60 s. When the the data was fitted with an adsorption-limited kinetic model, a forward rate constant of 5.03 × 105 s-1 was found.

Towards DNA methylation detection using biosensors.

DNA methylation, a stable and heritable covalent modification which mostly occurs in the context of a CpG dinucleotide, has great potential as a biomarker to detect disease, provide prognoses and predict therapeutic responses. It can be detected in a quantitative manner by many different approaches both genome-wide and at specific gene loci, in various biological fluids such as urine, plasma, and serum, which can be obtained without invasive procedures. The current, classical methods are effective in studying DNA methylation patterns, however, for the most part; they have major drawbacks such as expensive instruments, complicated and time consuming protocols as well as relatively low sensitivity, and high false positive rates. To overcome these obstacles, great efforts have been made toward the development of reliable sensor devices to solve these limitations, providing sensitive, fast and cost-effective measurements. The use of biosensors for DNA methylation biomarkers has increased in recent years, because they are portable, simple, rapid, and inexpensive which offers a straightforward way to detect methylated biomarkers. In this review, we give an overview of the conventional techniques for the detection of DNA methylation and then will focus on recent advances in biosensor based methylation detection that eliminate bisulfite conversion and PCR amplification.

DNA Origami Reorganizes upon Interaction with Graphite: Implications for High-Resolution DNA Directed Protein Patterning.

Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material.

Interactions of DNA with graphene and sensing applications of graphene field-effect transistor devices: a review.

Graphene field-effect transistors (GFET) have emerged as powerful detection platforms enabled by the advent of chemical vapor deposition (CVD) production of the unique atomically thin 2D material on a large scale. DNA aptamers, short target-specific oligonucleotides, are excellent sensor moieties for GFETs due to their strong affinity to graphene, relatively short chain-length, selectivity, and a high degree of analyte variability. However, the interaction between DNA and graphene is not fully understood, leading to questions about the structure of surface-bound DNA, including the morphology of DNA nanostructures and the nature of the electronic response seen from analyte binding. This review critically evaluates recent insights into the nature of the DNA graphene interaction and its affect on sensor viability for DNA, small molecules, and proteins with respect to previously established sensing methods. We first discuss the sorption of DNA to graphene to introduce the interactions and forces acting in DNA based GFET devices and how these forces can potentially affect the performance of increasingly popular DNA aptamers and even future DNA nanostructures as sensor substrates. Next, we discuss the novel use of GFETs to detect DNA and the underlying electronic phenomena that are typically used as benchmarks for characterizing the analyte response of these devices. Finally, we address the use of DNA aptamers to increase the selectivity of GFET sensors for small molecules and proteins and compare them with other, state of the art, detection methods.

From nonfinite to finite 1D arrays of origami tiles.

CONSPECTUS: DNA based nanotechnology provides a basis for high-resolution fabrication of objects almost without physical size limitations. However, the pathway to large-scale production of large objects is currently unclear. Operationally, one method forward is to use high information content, large building blocks, which can be generated with high yield and reproducibility. Although flat DNA origami naturally invites comparison to pixels in zero, one, and two dimensions and voxels in three dimensions and has provided an excellent mechanism for generating blocks of significant size and complexity and a multitude of shapes, the field is young enough that a single "brick" has not become the standard platform used by the majority of researchers in the field. In this Account, we highlight factors we considered that led to our adoption of a cross-shaped, non-space-filling origami species, designed by Dr. Liu of the Seeman laboratory, as the building block ideal for use in the fabrication of finite one-dimensional arrays. Three approaches that can be employed for uniquely coding origami-origami linkages are presented. Such coding not only provides the energetics for tethering the species but also uniquely designates the relative orientation of the origami building blocks. The strength of the coding approach implemented in our laboratory is demonstrated using examples of oligomers ranging from finite multimers composed of four, six, and eight origami structures to semi-infinite polymers (100mers). Two approaches to finite array design and the series of assembly steps that each requires are discussed. The process of AFM observation for array characterization is presented as a critical case study. For these soft species, the array images do not simply present the solution phase geometry projected onto a two-dimensional surface. There are additional perturbations associated with fluidic forces associated with sample preparation. At this time, reconstruction of the "true" or average solution structures for blocks is more readily achieved using computer models than using direct imaging methods. The development of scalable 1D-origami arrays composed of uniquely addressable components is a logical, if not necessary, step in the evolution of higher order fully addressable structures. Our research into the fabrication of arrays has led us to generate a listing of several important areas of future endeavor. Of high importance is the re-enforcement of the mechanical properties of the building blocks and the organization of multiple arrays on a surface of technological importance. While addressing this short list of barriers to progress will prove challenging, coherent development along each of these lines of inquiry will accelerate the appearance of commercial scale molecular manufacturing.

Rapid, high yield, directed addition of quantum dots onto surface bound linear DNA origami arrays.

We have developed an approach, which routinely generates ~10 micron long one dimensional (1D) arrays of DNA origami. Coupled with a sequential assembly method with a very short (~1 min) reaction time, this extended platform enables the production, in high yield, of 1D arrays of biomolecules or conjugates.

Site-specific immobilization of single-walled carbon nanotubes onto single and one-dimensional DNA origami.

Development of a simple and efficient methodology to control the placement, spacing, and alignment of single-walled carbon nanotubes (SWCNTs) is essential for nanotechnology device application. Building on the growing understanding that the strong π-π interaction between the bases of single-stranded DNA (ssDNA) and CNTs is sufficient not only to drive CNT solubility in water but also to stabilize individual nanotubes against clustering in aqueous solution, a new motif for functionalizing DNA origami (DO) with CNTs is demonstrated. CNTs solubilized via wrapping with ssDNA react with DO constructs displaying linear arrays of ssDNA, leading to immobilization of the CNTs onto the DO scaffold. This study demonstrates the immobilization of ssDNA-wrapped CNTs at specific positions on single DO constructs. Furthermore, multiple DO constructs assembled into extended one-dimensional arrays have been used to successfully align pairs of CNTs exceeding 500 nm in length in a parallel orientation. This result provides a simplified, alternative approach to immobilization of CNTs with programmed spacing and orientation.

Structural DNA nanotechnology: from design to applications.

The exploitation of DNA for the production of nanoscale architectures presents a young yet paradigm breaking approach, which addresses many of the barriers to the self-assembly of small molecules into highly-ordered nanostructures via construct addressability. There are two major methods to construct DNA nanostructures, and in the current review we will discuss the principles and some examples of applications of both the tile-based and DNA origami methods. The tile-based approach is an older method that provides a good tool to construct small and simple structures, usually with multiply repeated domains. In contrast, the origami method, at this time, would appear to be more appropriate for the construction of bigger, more sophisticated and exactly defined structures.

Thiolated dendrimers as multi-point binding headgroups for DNA immobilization on gold.

The synthesis of multithiolated DNA molecules that can be used to produce self-assembled monolayers of single-stranded DNA oligonucleotides on gold substrates is described. Generation 3 polyamidoamine (PAMAM) dendrimers were conjugated to DNA oligomers and functionalized with ~30 protected thiol groups. The protected thiol groups-thioacetate groups-allowed the dendrimer-DNA constructs to be stored in a buffer solution for at least 2 months before deprotection without any observable decrease in their ability to assemble into functional layers. The monolayers formed using these multithiolated DNA probe strands demonstrate target capture efficiencies comparable to those of analogous monolayers assembled with DNA functionalized with single thiol groups. A functional advantage of using dendrimer headgroups is the resistance to probe strand loss in prolonged exposure to buffer solutions at a high temperature (95 °C).

DNA origami impedance measurement at room temperature.

The frequency response of triangular DNA origami is obtained at room temperature. The sample shows a high impedance at low frequencies, e.g., at zero frequency 20 Gohms, which decreases almost linearly with the logarithm of the frequency reaching a low and flat value at 100 kHz where the impedance turns from capacitive to resistive, concluding that DNA can be used for transmission of signals at frequencies larger than 100 kHz. It is also found that characteristics of DNA cannot be completely disentangled from the characteristics of the substrate on which it is deposited, making the design of molecular circuits more challenging than the design of circuits with present lumped devices; this is a natural feature at the nanoscale.

Current-voltage-temperature characteristics of DNA origami.

The temperature dependences of the current-voltage characteristics of a sample of triangular DNA origami deposited in a 100 nm gap between platinum electrodes are measured using a probe station. Below 240 K, the sample shows high impedance, similar to that of the substrate. Near room temperature the current shows exponential behavior with respect to the inverse of temperature. Sweep times of 1 s do not yield a steady state; however sweep times of 450 s for the bias voltage secure a steady state. The thermionic emission and hopping conduction models yield similar barriers of approximately 0.7 eV at low voltages. For high voltages, the hopping conduction mechanism yields a barrier of 0.9 eV and the thermionic emission yields 1.1 eV. The experimental data set suggests that the dominant conduction mechanism is hopping in the range 280-320 K. The results are consistent with theoretical and experimental estimates of the barrier for related molecules.

NTA directed protein nanopatterning on DNA Origami nanoconstructs.

Precisely patterning proteins and other molecules at the nanoscale is crucial to future biosensing and optoelectronic applications. One- and two-dimensional DNA nanoconstructs have proven to be useful scaffolds for nanopatterning. This paper demonstrates the application of nitrilotriacetic acid (NTA) forming chelate complexes to localize histidine (His) tagged proteins via Ni(2+) ions onto DNA based structures. Particularly, enhanced green fluorescent protein (EGFP) was directed to specific surface locations on a designed DNA Origami nanoconstruct, and the resulting EGFP nanopattern was visualized using atomic force microscopy (AFM).

Impedance measurements on a DNA junction.

1 microm double-stranded DNA molecules are immobilized between pairs of gold and pairs of platinum microelectrodes with gaps of 0.4 and 1 microm, respectively, and their electrical characteristics are determined under the application of constant and sinusoidal bias voltages. Due to their extremely high impedance for constant voltage bias, the samples of DNA are excellent insulators; however, their impedances show strong frequency dependence in the range of 10 Hz-7.5 MHz. Favorable response in the gold electrodes is attributed to the higher ability of DNA molecules to bridge the narrower gold electrode gaps in contrast to that in the wider platinum junctions.

DNA attachment chemistry at the flexible silicone elastomer surface: toward disposable microarrays.

This paper describes the preparation and surface characterization of maleimide-activated silicone elastomer (PDMS(MCC)) followed by covalent functionalization using thiol-terminated DNA sequences (primary oligo). The stability of this attachment chemistry was demonstrated by the retention of the primary oligo through the process of hybridization with a labeled complementary DNA sequence. In these studies, the hybridized labeled DNA oligomers were detected using confocal fluorescence microscopy. We have employed a vapor deposition technique in which a plasma-treated silicone elastomer (PDMS(OH)) was exposed to vapors of 3-(aminopropyl)triethoxysilane (APTS) under vacuum, to yield the amine-functionalized silicone elastomer (PDMS(NH)(2)). PDMS(NH)(2) was further coupled with a heterofunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate to obtain PDMS(MCC). The surface functionalities of the elastomers were characterized using contact angle measurements and X-ray photoelectron spectroscopy. Surface-modified silicone elastomers appear to be promising substrates for use as substrates for disposable microarrays.