A Simple Colorimetric Assay of Bleomycin-Mediated DNA Cleavage Utilizing Double-Stranded DNA-Modified Gold Nanoparticles.

A colorimetric assay of DNA cleavage by bleomycin (BLM) derivatives was developed utilizing high colloidal stability on double-stranded (ds) DNA-modified gold nanoparticles (dsDNA-AuNPs) possessing a cleavage site. The assay was performed using dsDNA-AuNPs treated with inactive BLM or activated BLM (Fe(II)⋅BLM). A 10-min exposure in dsDNA-AuNPs with inactive BLM treatment resulted in a rapid color change from red to purple because of salt-induced non-crosslinking aggregation of dsDNA-AuNPs. In contrast, the addition of active Fe(II)⋅BLM retained the red color, probably because of the formation of protruding structures at the outermost phase of dsDNA-AuNPs caused by BLM-mediated DNA cleavage. Furthermore, the results of our model experiments indicate that oxidative base release and DNA-cleavage pathways could be visually distinguished with color change. The present methodology was also applicable to model screening assays using several drugs with different mechanisms related to antitumor activity. These results strongly suggest that this assay with a rapid color change could lead to simple and efficient screening of potent antitumor agents.

Suppressed DNA base pair stacking assembly of gold nanoparticles in an alcoholic solvent for enhanced ochratoxin A detection in Baijiu.

The currently established DNA nanoprobes for the detection of mycotoxin from beverages have been limited by complicated sample pretreatment and uncontrollable nanoparticle flocculation in complex systems. We develop a rapid colorimetric approach for ochratoxin A (OTA) detection in Baijiu in a sample-in/"yes" or "no" answer-out fashion through target-modulated base pair stacking assembly of DNA-functionalized gold nanoparticles (DNA-AuNPs). The colorimetric signification of OTA relies on the competition of OTA with the AuNP surface-grafted DNA in binding with an OTA-targeted aptamer. The specific recognition of OTA by the aptamer prevents DNA duplex formation on the AuNP surface, thereby inhibiting the base pair stacking assembly of the DNA-AuNPs and giving rise to a "turn-on" color. By further suppressing DNA hybridization using a bulged loop design and an alcohol solution, the DNA-AuNPs exhibit an improved reproducibility for OTA sensing while maintaining excellent susceptivity to OTA. A detection limit of 88 nM was achieved along with high specificity towards OTA, which is lower than the maximum tolerated level of OTA in foodstuffs defined by countries worldwide. The entire reaction time, avoiding sample pretreatment, is less than 17 min. The DNA-AuNPs with anti-interference features and sensitive "turn-on" performance promise convenient on-site detection of mycotoxin from daily beverages.

Generation of transmitochondrial cybrids using a microfluidic device.

Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs single mitochondrion transfer from a mtDNA-intact cell to a mtDNA-less (ρ0) cell by promoting cytoplasmic connection through a microtunnel between them. In the present study, we described a method for generating transmitochondrial cybrids using the microfluidic device. After achieving mitochondrial transfer between HeLa cells and thymidine kinase-deficient ρ0143B cells using the microfluidic device, selective culture was carried out using a pyruvate and uridine (PU)-absent and 5-bromo-2'-deoxyuridine-supplemented culture medium. The resulting cells contained HeLa mtDNA and 143B nuclei, but both 143B mtDNA and HeLa nuclei were absent in these cells. Additionally, these cells showed lower lactate production than parent ρ0143B cells and disappearance of PU auxotrophy for cell growth. These results suggest successful generation of transmitochondrial cybrids using the microfluidic device. Furthermore, we succeeded in selective harvest of generated transmitochondrial cybrids under a PU-supplemented condition by removing unfused ρ0 cells with puromycin-based selection in the microfluidic device.

G-Quadruplex-Functionalized Gold Nanoparticles for a Real-Time Biomolecule Sensor with On-Demand Tunable Properties.

G-quadruplex (G4) DNA-functionalized gold nanoparticles (AuNPs) were fabricated for a new sensing platform for a biomolecule, thrombin. Thrombin-binding aptamer (TBA), which forms a highly ordered G4 structure, was immobilized on AuNPs. The particles were induced to aggregate by binding of thrombin to G4 DNA. Thrombin was thus detected by the color change of the colloidal system from red to purple-blue. The aggregation was not due to the bridging between the particles through thrombin but to the reduction in steric repulsion attributable to the mobility and flexibility of G4 DNA. The change in the colloidal stability was quick and the bathochromic peak shift varied with the concentration of thrombin. The sensor showed a high specificity to the thrombin target over major proteins in human serum. The detection sensitivity and analytical performance were successfully tuned for an on-demand sensor with a linearity of 10.0-40.0 nM. The limits of detection and of quantification were 3.6 and 10.7 nM, respectively.

Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis.

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid-rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB-suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB-suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.

Terminal Sequence-Specific Interparticle Attraction between DNA Duplex-Carrying Polystyrene Microparticles in Aqueous Salt Solution Assessed by Optical Tweezers.

The dispersion behavior of DNA duplex-carrying colloidal particles in aqueous high-salt solutions shows extraordinary selectivity against the duplex terminal sequence. We investigated the interparticle force between DNA duplex-carrying polystyrene (dsDNA-PS) microparticles in aqueous salt solutions and examined their behavior in relation to the duplex terminal sequences. Force-distance (F-D) curves for a pair of dsDNA-PS particles were recorded with a dual-beam optical tweezers system with the two optically trapped particles closely approaching each other. Interestingly, only 3-5% of the oligo-DNA strands on the dsDNA-PS particles formed a duplex with complementary DNAs, and the F-D curves showed a distinct specificity to the duplex terminal sequences in the interparticle force at a high-NaCl concentration; a clear attraction peak was observed in F-D curves only when the duplex terminal was a complementary base pair. The attractive strength reached 2.6 ± 0.5 pN at 500 mM NaCl and 4.3 ± 1.0 pN at 750 mM NaCl. By sharp contrast, no significant attraction occurred for the particles with mismatched duplex terminals even at 750 mM NaCl. Similar duplex terminal-specificity in the interparticle force was also confirmed for dsDNA-PS particles in divalent MgCl2 solutions. Considering that the duplex terminal sequences on the dsDNA-PS particles showed only a negligible difference in their surface charges under identical salt conditions, we concluded that the interparticle attraction observed only for the dsDNA-PS particles with complementary duplex terminals is attributable to the salt-facilitated stacking interaction between the paired terminal nucleobases (i.e., blunt-end stacking) on the dsDNA-PS surfaces. Our results thus demonstrate the occurrence of a duplex terminal-specific interparticle force between dsDNA-PS particles under high-salt conditions.

Plasmon switching of gold nanoparticles through thermo-responsive terminal breathing of surface-grafted DNA in hydrated ionic liquids.

Self-assembly performed in ionic liquids (ILs) as a unique solvent promises distinct functions and applications in sensors, therapeutics, and optoelectronic devices due to the rich interactions between nanoparticle building blocks and ILs. However, the general consideration that common nanoparticles are readily destabilized by counterions in an IL has largely prevented researchers from investigating controlled nanoparticle assembly in IL-based systems. This study explores the assembling behaviour of double-stranded (ds) DNA-functionalized gold nanoparticles (dsDNA-AuNPs) in hydrated ionic liquids. The DNA base pair stacking assembly of dsDNA-AuNPs occurs at a low IL concentration (<5%). However, a moderate ionic liquid concentration (5-40%) can de-hybridize dsDNA and leaves single-stranded (ss) DNA stabilizing the AuNPs. In concentrated ionic liquids (>40%), interestingly, the higher ionic strength leads to the assembly of DNA-AuNPs. The triphasic assembly trend is also generally observed regardless of the type of IL. By down-regulation of DNA's melting temperature with the IL, the assembly of DNA-AuNPs affords robust response to a lower temperature range, promising applications in plasmonic devices and range-tunable temperature sensors.

DNA-Programmed Bimodal 2D Assembly of Differently Sized Nanoparticles via Folding of Precursory Circular Chains.

Gold nanoparticle (AuNP) assemblies in two-dimensions (2D) exhibit collective physical/chemical properties that are useful for various devices. However, technical issues still impede the efficient ordering of differently sized AuNPs on solid supports while avoiding phase separation. This paper describes a method to construct binary 2D assemblies by folding precursory circular chains composed of small and large AuNPs. The structural change is caused by a spontaneous, non-cross-linking assembly of fully matched double-stranded DNA-modified AuNPs (dsDNA-AuNPs) at a high ionic strength. Since larger dsDNA-AuNPs have a lower critical coagulation concentration of the supporting electrolyte, the spontaneous assembly of large AuNPs precedes that of small AuNPs in the precursory chain during evaporation. Transmission electron microscopy reveals that alternate-type AuNP chains are folded into a binary 2D structure in a mixed mode, whereas block-type chains are transformed into a binary 2D structure in a core-shell mode. The methodology could potentially be harnessed for the fabrication of binary AuNP arrays for various devices.

Accelerated non-crosslinking assembly of DNA-functionalized nanoparticles in alcoholic solvents: for application in the identification of clear liquors.

Colorimetric detection of various target molecules in aqueous solutions based on the non-crosslinking assembly of DNA-functionalized Au nanoparticles (DNA-AuNPs) has been well established in recent years. The extension of DNA-AuNPs to other solvents remains much less explored, despite the practical importance of detection in non-aqueous solutions, such as those containing an organic ingredient that is required or not removable in many contexts. However, the general consideration that DNA is easily denatured and precipitated in organic solvents has been hampering the use of DNA-AuNPs in low polar solvents. Herein, we report a more rapid non-crosslinking assembly of double-stranded (ds) DNA-AuNPs in alcoholic solvents than in aqueous solvents. When the concentration of ethanol in the disperse medium is increased from 0% to 20% (v/v), the rate of non-crosslinking assembly is distinctly increased by a factor of 5-6, whereas the rate is sharply decreased when the ethanol concentration is further increased to 40%. This biphasic kinetics trend could be attributed to the competitive balance between the enhanced intermolecular attraction between dsDNAs and the increased propensity for melting of dsDNA. Rapid naked-eye identification of clear liquors that are encoded by oligonucleotide additives has also been demonstrated by using the alcoholic non-crosslinking assembly of dsDNA-AuNPs as a proof-of-concept.

Cytoplasmic fusion between an enlarged embryonic stem cell and a somatic cell using a microtunnel device.

Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed. However, the ES cell was too small for cell pairing at the device. Therefore, in the present study, ES cell enlargement was carried out with the colchicine derivative demecolcine (DC). DC induced enlargement of ES cells without loss of their stemness. When an enlarged ES cell was paired with a somatic cell in the microtunnel device, cytoplasmic fusion was observed. The present method may be useful for further development of reprogramming techniques for iPS cell preparation without gene transfection.

Chemically Fueled Plasmon Switching of Gold Nanorods by Single-Base Pairing of Surface-Grafted DNA.

The interactions between metal ions and biomolecules are crucial to various bioprocesses. Development of plasmon switching nanodevices that exploit these molecular interactions is of fundamental and technological interest. Here, we show plasmon switching based on rapid aggregation/dispersion of double-stranded DNA-modified gold nanorods (dsDNA-AuNRs) that exhibit colloidal behaviors depending on pairing/unpairing of the terminal bases. The dsDNA-AuNRs bearing a thymine-thymine (T-T) mismatch at the penultimate position undergo spontaneous non-cross-linking aggregation in the presence of Hg2+ due to T-Hg-T base pairing. Inversely, the subsequent addition of cysteine (Cys) gives rise to the removal of Hg2+ from the T-Hg-T base pair to reproduce the T-T mismatch, resulting in stable dispersion of the dsDNA-AuNRs. The chemical-responsive plasmon switch allows for the rapid and repeatable cycles at room temperature. The validity of the present method is further exemplified by developing another plasmon switch fueled by Ag+ and Cys by installing the Ag+-binding DNA sequence in the dsDNA-AuNR.

Characterizing the non-crosslinked aggregation of DNA-modified gold nanoparticles: effects of DNA length and terminal base pair.

We previously reported that fully complementary DNA duplexes formed on gold nanoparticle (GNP) surfaces aggregate at high salt concentrations. We previously reported that DNA-functionalized gold nanoparticles (GNPs) aggregate by hybridization with fully complementary DNA at high salt concentrations. Although this behavior has been applied to some precise naked-eye colorimetric analyses of DNA-related molecules, the aggregation mechanism is still unclear and comprehensive studies are needed. In this paper, we reveal the key factors that influence GNP aggregation. The effects of temperature, electrolyte concentration, probe length, and particle size, which control the stabilities of double-stranded DNAs and GNPs, were investigated. Larger GNPs aggregated more easily, and GNP aggregates were easily formed with ∼15-mer-long probes, while longer probes prevented aggregation, perhaps by preventing the formation of rigid double-stranded DNA layers, compared to shorter probes. Furthermore, GNPs with purine bases at their 5' ends aggregated more easily than those with these bases at their 3' ends. This phenomenon is different from that based on the melting-temperature trend calculated using the nearest-neighbor method.

Toxicity of insulin-derived amyloidosis: a case report.

BACKGROUND: Insulin-derived amyloidosis is a skin-related complication of insulin therapy that interferes with insulin therapy. Although toxicities of in vitro-formed insulin amyloid fibrils have been well studied, the toxicity of insulin-derived amyloidosis remains to be clarified. CASE PRESENTATION: A 58-year-old man with type 2 diabetes mellitus underwent a lower limb amputation due to diabetic gangrene. Several antibiotics including minocycline were administered for infection and sepsis. A hard mass at the insulin injection sites in the lower abdomen was discovered by chance four months later. Although no abnormal findings in the surface skin of the mass were observed, necrotic tissue was seen around the mass when a biopsy was performed. Histological and toxicity studies were performed for this patient and four other patients with abdominal masses at insulin injection sites. Histological and immunohistochemical studies showed that the masses had typical characteristics of amyloid deposits in all cases, whereas necrotic findings were seen adjacent to the amyloid deposit only in the case presented. Toxicity studies indicated that the amyloid tissue from the present case had significant cell toxicity compared to the control skin tissue or the amyloid tissues from the other four cases. CONCLUSIONS: This report showed that toxic insulin-derived amyloidosis can occur. In addition, this report suggested that toxic insulin-derived amyloidosis may cause necrosis in the surrounding tissue. Although the toxic amyloid deposit of insulin-derived amyloidosis was found in only one patient, no structural differences between toxic and non-toxic deposits were seen on histological and immunohistochemical studies.

Non-Cross-Linking Aggregation of DNA-Carrying Polymer Micelles Triggered by Duplex Formation.

Colloidal behaviors of particles functionalized with biomolecules are generally complicated. This study describes that colloidal behaviors of double-stranded (ds) DNA-carrying polymer micelles are well controlled by altering the molar ratio of single-stranded (ss) DNA moiety in the dsDNA shell. ssDNA-carrying micelles composed of a poly( N-isopropylacrylamide) (PNIPAAm) core surrounded by a dense shell of ssDNAs were prepared through self-assembly of PNIPAAm grafted with ssDNA by incubating its solution above the lower critical solution temperature. Spontaneous, non-cross-linking aggregation of the micelles was triggered by DNA duplex formation on the surface. Comparison of the critical coagulation concentration of NaCl among a series of the DNA-carrying micelles revealed the relationship between the helical structure of the surface-bound DNA and the colloidal stability of the micelles. The electrophoretic mobility analysis of the micelles indicated that the duplex formation reduced the structural flexibility of the surface-bound DNA, thereby decreasing the interparticle entropic repulsion. It is also suggested that the augmented rigidity of the surface-bound DNA increases the number of terminal base pairs facing the solvent, which could lead to multiple blunt-end stacking interaction among the micelles. Therefore, small DNA molecules could be considered unique surface-modifiers capable of controlling interactions between the surfaces of materials.

Stochastic Binding Process of Blunt-End Stacking of DNA Molecules Observed by Atomic Force Microscopy.

Hydrophobic attraction is often a physical origin of nonspecific and irreversible (uncontrollable) processes observed for colloidal and biological systems, such as aggregation, precipitation, and fouling with biomolecules. On the contrary, blunt-end stacking of complementary DNA duplex chain pairs, which is also mainly driven by hydrophobic interaction, is specific and stable enough to lead to self-assemblies of DNA nanostructures. To understand the reason behind these contradicting phenomena, we measured forces operating between two self-assembled monolayers of duplexed DNA molecules with blunt ends (DNA-SAMs) and analyzed their statistics. We found the high specificity and stability of blunt-end stacking that resulted in the high resemblance between the interaction forces measured on approaching and retracting. The other finding is on the stochastic formation process of blunt-end stacking, which appeared as a significant fluctuation of the interaction forces at separations smaller than 2.5 nm. Based on these results, we discuss the underlying mechanism of the specificity and stability of blunt-end stacking.

Directed Assembly of Gold Nanorods by Terminal-Base Pairing of Surface-Grafted DNA.

Directed assemblies of anisotropic metal nanoparticles exhibit attractive physical and chemical properties. However, an effective methodology to prepare differently directed assemblies from the same anisotropic nanoparticles is not yet available. Gold nanorods (AuNRs) region-selectively modified with different DNA strands can form side-by-side (SBS) and end-to-end (ETE) assemblies in a non-crosslinking manner. When the complementary DNA is hybridized to the surface-bound DNA, stacking interaction between the blunt ends takes place in the designated regions. Such AuNRs assemble into highly ordered structures, assisted by capillary forces emerging on the substrate surface. Moreover, insertion of a mercury(II)-mediated thymine-thymine base pair into the periphery of the DNA layer allows selective formation of the SBS or ETE assemblies from the strictly identical AuNRs with or without mercury(II).

Cross-Linking versus Non-Cross-Linking Aggregation of Gold Nanoparticles Induced by DNA Hybridization: A Comparison of the Rapidity of Solution Color Change.

Gold nanoparticles densely modified with single-stranded DNA (ssDNA-AuNPs) form aggregates with cross-linker ssDNAs via duplex formation. Alternatively, the ssDNA-AuNPs are spontaneously aggregated at high ionic strength in a non-cross-linking manner when complementary ssDNAs are added to form fully matched duplexes. Both aggregation modes are accompanied by a red-to-purple color change, which has been exploited in various bioassays. The current study compares the rapidity of color change between the cross-linking and non-cross-linking aggregation modes under identical conditions. When a small number of cross-linker/complementary DNAs are provided, the cross-linking mode exhibited more rapid color change than the non-cross-linking mode. Conversely, with a large number of the DNAs, the non-cross-linking aggregation occurred more rapidly than the cross-linking counterpart. This finding allows one to select a more appropriate aggregation mode for application of ssDNA-AuNPs to colorimetric assays under given conditions.

Rapid Non-Crosslinking Aggregation of DNA-Functionalized Gold Nanorods and Nanotriangles for Colorimetric Single-Nucleotide Discrimination.

Gold nanoparticles modified with DNA duplexes are rapidly and spontaneously aggregated at high ionic strength. In contrast, this aggregation is greatly suppressed when the DNA duplex has a single-base mismatch or a single-nucleotide overhang located at the outermost surface of the particle. These colloidal features emerge irrespective of the size and composition of the particle core; however, the effects of the shape remain unexplored. Using gold nanorods and nanotriangles (nanoplatelets), we show herein that both remarkable rapidity in colloidal aggregation and extreme susceptibility to DNA structural perturbations are preserved, regardless of the shape and aspect ratio of the core. It is also demonstrated that the DNA-modified gold nanorods and nanotriangles are applicable to naked-eye detection of a single-base difference in a gene model. The current study corroborates the generality of the unique colloidal properties of DNA-functionalized nanoparticles, and thus enhances the feasibility of their practical use.

Cryopreservation of adhered mammalian cells on a microfluidic device: Toward ready-to-use cell-based experimental platforms.

In this paper, we describe cryopreservation of mammalian cells in the adhered state on a microfluidic device (microdevice) for the first time. HeLa, NIH3T3, MCF-7, and PC12 cells were cultured on a microdevice in which a commercial polystyrene dish surface was used as the cell adhesion surface. Without cell-detaching treatment, the microdevice was stored in a freezer at -80°C. After thawing, we observed a greater number of live cells on the microdevice than those on a control culture dish. Although the effectiveness of the microdevice varied depending on the cell type and surface coating, the trend was consistent. We confirmed that the phenotype of the PC12 cells to differentiate into neuron-like cells was kept after the on-chip cryopreservation, and that the results of cytotoxicity test of cisplatin against the HeLa cells were essentially unchanged by the on-chip cryopreservation. These findings will open up a new possibility of ready-to-use cell-based experimental platforms.

Effects of Complementary DNA and Salt on the Thermoresponsiveness of Poly(N-isopropylacrylamide)-b-DNA.

The thermoresponsive structural transition of poly(N-isopropylacrylamide) (PNIPAAm)-b-DNA copolymers was explored. Molecular assembly of the block copolymers was facilitated by adding salt, and this assembly was not nucleated by the association between DNA strands but by the coil-globule transition of PNIPAAm blocks. Below the lower critical solution temperature (LCST) of PNIPAAm, the copolymer solution remained transparent even at high salt concentrations, regardless of whether DNA was hybridized with its complementary partner to form a double-strand (or single-strand) structure. At the LCST, the hybridized copolymer assembled in spherical nanoparticles, surrounded by double-stranded DNA; subsequently, the non-cross-linking aggregation occurred, while the nanoparticles were dispersed if the salt concentration was low or DNA blocks were unhybridized. When the DNA duplex was denatured to a single-stranded state by heating, the aggregated nanoparticles redispersed owing to the recovery of the steric repulsion of the DNA strands. The changes in the steric and electrostatic effects by hybridization and the addition of salt did not result in any specific attraction between DNA strands but merely decreased the repulsive interactions. The van der Waals attraction between the nanoparticles overcame such repulsive interactions so that the non-cross-linking aggregation of the micellar particles was mediated.