Heterotypic cell-in-cell structures between cancer and NK cells are associated with enhanced anticancer drug resistance.

The heterotypic CIC structures formed of cancer and immune cells have been observed in tumor tissues. We aimed to assess the feasibility of using heterotypic CICs as a functional biomarker to predict NK susceptibility and drug resistance. The heterotypic CIC-forming cancer cells showed a lower response to NK cytotoxicity and higher proliferative ability than non-CIC cancer cells. After treatment with anticancer drugs, cancer cells that formed heterotypic CICs showed a higher resistance to anticancer drugs than non-CIC cancer cells. We also observed the formation of more CIC structures in cancer cells treated with anticancer drugs than in the non-treated group. Our results confirm the association between heterotypic CIC structures and anticancer drug resistance in CICs formed from NK and cancer cells. These results suggest a mechanism underlying immune evasion in heterotypic CIC cancer cells and provide insights into the anticancer drug resistance of cancer cells.

Fabrication of electrochemical biosensor composed of multi-functional DNA structure/Au nanospike on micro-gap/PCB system for detecting troponin I in human serum.

Acute myocardial infarction (AMI) is one of the most serious diseases affecting human beings. In this study, in order to rapidly detect AMI disease, the authors fabricated a label-free electrochemical biosensor composed of a multi-functional DNA structure on Au nanospike (AuNS) with a fabricated Au micro-gap electrode which was incorporated with a PCB chip in order to detect cardiac troponin I (cTnI). As a bioprobe, the DNA 3 way-junction (3WJ) was introduced, because the DNA 3WJ has three arms for embodying the multi-functionality. Each piece of DNA was assembled to simultaneously form the DNA 3WJ for cTnI detection, signal transduction, and immobilization, respectively. The assembled DNA 3WJ structure was confirmed by Native-TBM PAGE. Moreover, in order to increase the electrochemical signal sensitivity, AuNS was prepared. The Au micro-gap array is fabricated with a printed circuit board (PCB) chip in order to control each micro-gap electrode panel selectively so as to detect low volumes of cTnI. Then, the DNA strucuture on pAuNS-modified electrode was prepared using the layer-by-layer (LbL) assembly method. FE-SEM and AFM were used to investigate the modified-surface morphology. The cyclic voltammetry (CV) was measured to confirm the cTnI binding to DNA 3WJ-modified electrode. cTnI was detected in the HEPES solution and human serum, respectively. The LOD result exhibited 1.0 pM in HEPES solution and 1.0 pM in 20% diluted human serum, respectively. In addition, the selectivity test was carried out with various proteins as the control experiment. The present study showed label-free, simple fabrication, and easy-to-tailor detection elements for cTnI.

Electrochemical detection of dopamine using periodic cylindrical gold nanoelectrode arrays.

Dopamine is a key molecule in neurotransmission and has been known to be responsible for several neurological diseases. Hence, its sensitive and selective detection is important for the early diagnosis of diseases related to abnormal levels of dopamine. In this study, we reported a new cylindrical gold nanoelectrode (CAuNE) platform fabricated via sequential laser interference lithography and electrochemical deposition. Among the fabricated electrodes, CAuNEs with a diameter of 700 nm, 150 s deposited, was found to be the best for electrochemical dopamine detection. According to cyclic voltammetry results, the linear range of the CAuNE-700 nm was 1-100 µM of dopamine with a limit of detection (LOD) of 5.83 µM. Moreover, owing to the homogeneous periodic features of CAuNEs, human neural cells were successfully cultured and maintained for more than 5 days in vitro without the use of any extracellular matrix proteins and dopamine was detectable in the presence of these cells on the electrode. Therefore, we concluded that the developed dopamine sensing platform CAuNE can be used for many applications including early diagnosis of neurological diseases; function tests of dopaminergic neurons derived from various stem cell sources; and toxicity assessments of drugs, chemicals, and nanomaterials on human neuronal cells.

Recent developments of nano-structured materials as the catalysts for oxygen reduction reaction.

Developments of high efficient materials for electrocatalyst are significant topics of numerous researches since a few decades. Recent global interests related with energy conversion and storage lead to the expansion of efforts to find cost-effective catalysts that can substitute conventional catalytic materials. Especially, in the field of fuel cell, novel materials for oxygen reduction reaction (ORR) have been noticed to overcome disadvantages of conventional platinum-based catalysts. Various approaching methods have been attempted to achieve low cost and high electrochemical activity comparable with Pt-based catalysts, including reducing Pt consumption by the formation of hybrid materials, Pt-based alloys, and not-Pt metal or carbon based materials. To enhance catalytic performance and stability, numerous methods such as structural modifications and complex formations with other functional materials are proposed, and they are basically based on well-defined and well-ordered catalytic active sites by exquisite control at nanoscale. In this review, we highlight the development of nano-structured catalytic materials for ORR based on recent findings, and discuss about an outlook for the direction of future researches.

Three-Dimensional Graphene-RGD Peptide Nanoisland Composites That Enhance the Osteogenesis of Human Adipose-Derived Mesenchymal Stem Cells.

Graphene derivatives have immense potential in stem cell research. Here, we report a three-dimensional graphene/arginine-glycine-aspartic acid (RGD) peptide nanoisland composite effective in guiding the osteogenesis of human adipose-derived mesenchymal stem cells (ADSCs). Amine-modified silica nanoparticles (SiNPs) were uniformly coated onto an indium tin oxide electrode (ITO), followed by graphene oxide (GO) encapsulation and electrochemical deposition of gold nanoparticles. A RGD-MAP-C peptide, with a triple-branched repeating RGD sequence and a terminal cysteine, was self-assembled onto the gold nanoparticles, generating the final three-dimensional graphene-RGD peptide nanoisland composite. We generated substrates with various gold nanoparticle-RGD peptide cluster densities, and found that the platform with the maximal number of clusters was most suitable for ADSC adhesion and spreading. Remarkably, the same platform was also highly efficient at guiding ADSC osteogenesis compared with other substrates, based on gene expression (alkaline phosphatase (ALP), runt-related transcription factor 2), enzyme activity (ALP), and calcium deposition. ADSCs induced to differentiate into osteoblasts showed higher calcium accumulations after 14-21 days than when grown on typical GO-SiNP complexes, suggesting that the platform can accelerate ADSC osteoblastic differentiation. The results demonstrate that a three-dimensional graphene-RGD peptide nanoisland composite can efficiently derive osteoblasts from mesenchymal stem cells.

Investigation of Hemoglobin/Gold Nanoparticle Heterolayer on Micro-Gap for Electrochemical Biosensor Application.

In the present study, we fabricated a hemoglobin/gold nanoparticle (Hb/GNP) heterolayer immobilized on the Au micro-gap to confirm H2O2 detection with a signal-enhancement effect. The hemoglobin which contained the heme group catalyzed the reduction of H2O2. To facilitate the electron transfer between hemoglobin and Au micro-gap electrode, a gold nanoparticle was introduced. The Au micro-gap electrode that has gap size of 5 µm was fabricated by conventional photolithographic technique to locate working and counter electrodes oppositely in a single chip for the signal sensitivity and reliability. The hemoglobin was self-assembled onto the Au surface via chemical linker 6-mercaptohexanoic acid (6-MHA). Then, the gold nanoparticles were adsorbed onto hemoglobin/6-MHA heterolayers by the layer-by-layer (LbL) method. The fabrication of the Hb/GNP heterolayer was confirmed by atomic force microscopy (AFM) and surface-enhanced Raman spectroscopy (SERS). The redox property and H2O2 detection of Hb/GNP on the micro-gap electrode was investigated by a cyclic voltammetry (CV) experiment. Taken together, the present results show that the electrochemical signal-enhancement effect of a hemoglobin/nanoparticle heterolayer was well confirmed on the micro-scale electrode for biosensor applications.

Control of electrochemical signals from quantum dots conjugated to organic materials by using DNA structure in an analog logic gate.

Various bio-logic gates have been studied intensively to overcome the rigidity of single-function silicon-based logic devices arising from combinations of various gates. Here, a simple control tool using electrochemical signals from quantum dots (QDs) was constructed using DNA and organic materials for multiple logic functions. The electrochemical redox current generated from QDs was controlled by the DNA structure. DNA structure, in turn, was dependent on the components (organic materials) and the input signal (pH). Independent electrochemical signals from two different logic units containing QDs were merged into a single analog-type logic gate, which was controlled by two inputs. We applied this electrochemical biodevice to a simple logic system and achieved various logic functions from the controlled pH input sets. This could be further improved by choosing QDs, ionic conditions, or DNA sequences. This research provides a feasible method for fabricating an artificial intelligence system.

A biomemory chip composed of a myoglobin/CNT heterolayer fabricated by the protein-adsorption-precipitation-crosslinking (PAPC) technique.

In this study, a biomemory chip consisting of a myoglobin/carbon nanotube (CNT) heterolayer is fabricated via the protein-adsorption-precipitation-crosslinking (PAPC) technique for electrochemical signal enhancement, long-term stability, and improved memory function. The PAPC technique is used to fabricate a myoglobin/CNT heterolayer with a CNT core and a high-density myoglobin-shell structure to achieve efficient heterolayer formation and improved performance of the heterolayer. The fabricated myoglobin/CNT heterolayer is immobilized onto a Au substrate through a chemical linker. The surface morphology of the deposited heterolayer is investigated via transmission electron microscopy and atomic force microscopy. The redox properties of the myoglobin/CNT heterolayer are investigated by cyclic voltammetry, and the memory function of the heterolayer, including the "write step" and "erase step," is measured by chronoamperometry. Compared with the myoglobin monolayer without CNT, the myoglobin/CNT heterolayer fabricated by the PAPC technique exhibits greater electrochemical signal enhancement, long-term stability at room temperature, and improved memory function. The results suggest that the proposed myoglobin/CNT heterolayer produced via the PAPC technique can be applied as a platform for bioelectronic devices to achieve improved signal intensity and durability.

Electrochemical Bioelectronic Device Consisting of Metalloprotein for Analog Decision Making.

We demonstrate an analog type logical device that combines metalloprotein and organic/inorganic materials and can make an interactive analog decision. Myoglobin is used as a functional biomolecule to generate electrochemical signals, and its original redox signal is controlled with various mercapto-acids by the distance effect between myoglobin and a metal surface in the process of electron transfer. Controlled signals are modulated with the introduction of inorganic materials including nanoparticles and metal ions. By forming a hybrid structure with various constituents of organic/inorganic materials, several functions for signal manipulation were achieved, including enhancement, suppression, and shift. Based on the manipulated signals of biomolecules, a novel logical system for interactive decision-making processes is proposed by selectively combining different signals. Through the arrangement of various output signals, we can define interactive logical results regulated by an inherent tendency (by metalloprotein), personal experience (by organic spacer sets), and environments (by inorganic materials). As a practical application, a group decision process is presented using the proposed logical device. The proposed flexible logic process could facilitate the realization of an artificial intelligence system by mimicking the sophisticated human logic process.

Fatigue Test of Cytochrome C Self-Assembled on a 11-MUA Layer Based on Electrochemical Analysis for Bioelectronic Device.

A cytochrome c/11-MUA heterolayer was fabricated to analyze its electrochemical characteristics in harsh conditions for a stable bioelectronic device. Since a cytochrome c/11-MUA heterolayer has been applied to construct the bioelectronics device such as non-volatile biomemory device, an understanding of electrochemical property of the heterolayer in harsh conditions such as variation of temperature and pH, and repetition of usage is necessary to manufacture a stable platform of bioelectronic device. Cytochrome c, a metalloprotein to have a heme group, was self-assembled on the Au surface via the chemical linker 11-mercaptoundecanoic acid (11-MUA). Immobilization of the heterolayer was confirmed by surface-enhanced Raman spectroscopy (SERS) and scanning tunneling microscopy (STM). The fatigue test was done by investigating the redox properties based on cyclic voltammetry (CV) of the heterolayer. The retention time test and pH dependence, thermal test of the fabricated heterolayer were conducted by CV, which showed that the fabricated film retained redox properties for more than 33 days, and from pH 5.0 to pH 9.0, from 15 °C to 55 °C. Taken together, our results show that a cytochrome c/11-MUA heterolayer is very stable, which could be used as a platform of bioelectronic device.

Fabrication of new single cell chip to monitor intracellular and extracellular redox state based on spectroelectrochemical method.

Probing the local environment of target cells has been considered a challenging task due to the complexity of living cells. Here, we developed new single cell-based chip to investigate the intracellular and extracellular redox state of PC12 cells using spectroelectrochemical tool that combined surface-enhanced Raman scattering (SERS) and linear sweep voltammetry (LSV) techniques. PC12 cells immobilized on gold nanodots/ITO surface were subjected to LSV and their intracellular biochemical changes were successfully monitored by SERS simultaneously. Moreover, paired gold microelectrodes with micrometer-sized gap containing hexagonal array of gold nanodots were fabricated to detect electrochemical activity and changes in the redox environment of single PC12 cell based on SERS-LSV tool. This showed very effective detecting method. The used technology included the utilization of gold nanodots array inside micro-gap to enhance the Raman signals and the electrochemical activity of single cell. This could be used as an effective research tool to analyze cellular processes.

Electrochemical-signal enhanced information storage device composed of cytochrome c/SNP bilayer.

The films organized with biomolecules and organic materials are important elements for developing bioelectronic devices according to their electron transfer property. Until now, several concepts of techniques have been accomplished to be used for developing biomemory devices. However it is difficult to detect the current signal from the electron transfer between biomolecules and the substrate in these fabricated films. To enhance the current signal, the silver nanoparticle was introduced to the cytochrome c in this present study. The surface morphology of the fabricated film was investigated by atomic force microscopy. The current signal enhancement was investigated by cyclic voltammetry. As a result, we could obtain the redox potentials. Moreover, by chronoamperometry, we validated that this proposed layer showed the signal-enhanced memory property for biomemory devices. This new film composed of the cytochrome c and the silver nanoparticle showed the signal enhancement. Using chronoamperometry, the areas under the graphs between 0 s and 50 ms were calculated. The calculated result showed that the areas under the cytochrome c/SNP graph and cytochrome c graph were 6.93 x 10(-7) C and 4.54 x 10(-7) C, respectively. This numerical value verified that the cytochrome c/silver nanoparticle hetero-layer film showed better electron charged biomemory performance compared to the cytochrome c monolayer. This signal-enhanced film can be applied to the bioelectronic devices which are able to replace existing electronic devices in the near future.

An electrochemical H2O2 detection method based on direct electrochemistry of myoglobin immobilized on gold deposited ITO electrode.

A protein based electrochemical sensor for the detection of hydrogen peroxide based on Myoglobin immobilized on gold nano structures patterned on Indium tin oxide electrode was developed. A uniformly distributed nanometer sized Au-array on ITO electrode surface was obtained by optimizing electro deposition conditions. The morphology of Mb molecules and Au-nanostructures on ITO was investigated by scanning electron microscopy. A Cyclic voltammetry technique was employed to study electrochemical behavior of immobilized Mb on Au/ITO electrode. From CV, a pair of quasi-reversible redox peaks of Mb obtained in 10 mM PBS buffer solution at 0.28 and 0.11 V respectively. From the electrochemical experiments, it is observed that Mb/Au/ITO electrode provides a facile electron transfer between Mb and modified ITO electrode and it also catalyzes the reduction of H2O2. A linear increase in amperometric current with increase in H2O2 concentration was also observed. The stability, reusability and selectivity of the biosensor were also evaluated. The proposed biosensor exhibits an effective and fast catalytic response to reduction of H2O2 which can be used in future biosensor applications.

Nanoscale biomemory composed of recombinant azurin on a nanogap electrode.

We fabricate a nanoscale biomemory device composed of recombinant azurin on nanogap electrodes. For this, size-controllable nanogap electrodes are fabricated by photolithography, electron beam lithography, and surface catalyzed chemical deposition. Moreover, we investigate the effect of gap distance to optimize the size of electrodes for a biomemory device and explore the mechanism of electron transfer from immobilized protein to a nanogap counter-electrode. As the distance of the nanogap electrode is decreased in the nanoscale, the absolute current intensity decreases according to the distance decrement between the electrodes due to direct electron transfer, in contrast with the diffusion phenomenon of a micro-electrode. The biomemory function is achieved on the optimized nanogap electrode. These results demonstrate that the fabricated nanodevice composed of a nanogap electrode and biomaterials provides various advantages such as quantitative control of signals and exclusion of environmental effects such as noise. The proposed bioelectronics device, which could be mass-produced easily, could be applied to construct a nanoscale bioelectronics system composed of a single biomolecule.

Nanoscale biofilm modification-method concerning a myoglobin/11-MUA bilayers for bioelectronic device.

We developed surface modification tools for the fabrication of a bioelectronic device which consists of a myoglobin monolayer self-assembled on an 11-MUA layer. To utilize a single protein as the active element, it was necessary to reduce protein aggregation on the protein layer in the nanobio electronic device, which was developed in our previous study and shown to display basic biomemory functions. Here, the reduction of myoglobin aggregation was accomplished by using 3-(3-cholamidopropyl) dimethylammonio-11-propanesulfonate (CHAPS) to fabricate a well-defined protein layer on the bioelectronic device. We investigated two different surface modification methods for making well oriented biofilm. The effects of CHAPS on the formation of a myoglobin layer self-assembled on an 11-MUA layer were examined by atomic force microscopy and Raman spectroscopy. The size of the myoglobin aggregates was reduced from 200-250 nm to 10-40 nm depending on treatment method. The sustaining redox property of the CHAPS treated myoglobin layer was examined using cyclic voltammetry. Using these techniques, we found that after surfactant CHAPS treatment, protein aggregation was dramatically reduced and the protein layer still maintained its inherent electrochemical properties.

Control of nanogap separation by surface-catalyzed chemical deposition.

The nanogap devices, which comprise multiple electrodes separated by a few to a few tens of nanometers, have opened up new possibilities in biomolecular sensing as well as various frontier electronics. One of the key aspects of the nanogap device research is how to control the gap distance following each specific needs of the gap structure. Here, we report the extensive study on the fine control of the gap distance between electrodes within the range of 1-80 nm via surface-catalyzed chemical deposition. The initial gap electrodes were prepared via conventional e-beam lithography, and the gap distance was narrowed to a designed value through the surface-catalyzed reduction of gold ion on the predefined electrode surfaces, by simple dipping of the electrodes into the aqueous solution of gold chloride and hydroxylamine. The final gap distance was controlled by adjusting the repetition number, reductant concentration, reaction time, and reaction temperature. The dependence of the gap-narrowing reaction on these parameters was systematically examined based on the results of field emission scanning electron microscopy and atomic-force microscopy.

Fabrication of biofilm in nanoscale consisting of cytochrome f/2-MAA bilayer on Au surface for bioelectronic devices by self-assembly technique.

We developed the nanoscale biofilm consisting of cytochrome f self-assembled on 2-MAA layer to apply bioelectronic devices. As cytochrome f has redox property, it can be possible to apply bioelectronic devices. The fabricated biofilm was confirmed by SPR and STM experiment. And the electrochemical property was checked by CV, CA, and STS.

Nanoscale fabrication of myoglobin monolayer on self-assembled DTSSP for bioelectronic device.

The fabrication method of nanoscale myoglobin monolayer using chemical linker is introduced in this study because control of amount and orientation of protein immobilized on electronic device is one of main issues to be solved for the realization of biomolecular electronic device. Myoglobin, metalloprotein, is selected as active material due to its electrochemical property. To immobilize myoglobin on Au surface, 3,3-dithiobis (sulphosuccinimidyl propionate) (DTSSP) is utilized as a chemical linker. The optimum amount of protein is investigated by surface plasmon resonance (SPR). SPR and scanning tunneling microscope (STM) results confirm the nano scale protein layer formed on DTSSP self assembled monolayer (SAM) on Au surface. Protein layer on Au surface using DTSSP as chemical linker was more stable than random adsorption without linker as aspect of redox character due to the fact that myoglobin immobilized with chemical linker did not lose its redox property after long usages.

Investigation of the redox property of a metalloprotein layer self-assembled on various chemical linkers.

Myogloblin, a well-known metalloprotein, was immobilized on a gold surface using various chemical linkers to investigate the length effect of chemical linker on the electron transfer in protein layers, because chemical linkers play roles in the pathway that transfers the electron from the protein to the gold substrate and act as protein immobilization reagents. Chemical linkers with 2, 6, 11, and 16 carbons were utilized to confirm length-effects. The immobilization of protein and chemical linker was validated with surface plasmon resonance (SPR) and atomic force microscopy (AFM). The electrochemical property was evaluated by cyclic voltammetry (CV) and chronocoulometry (CC). In those results, redox peaks of immobilized protein were controlled via the length of chemical linkers, and it could be directly applied to the realization of bioelectronic device.

Synthesis and cytotoxic activity of 1-(1-benzoylindoline-5-sulfonyl)-4-phenylimidazolidinones.

The novel 1-(1-benzoylindoline-5-sulfonyl)-4-phenyl-4,5-dihydroimidazolones 2 shows highly potent and broad cytotoxicities. Their cytotoxicities against human lung carcinoma A549, human chronic myelogenous leukemia K562, and human ovarian adenocarcinoma SK-OV-3 are compatible with doxorubicin. Compound 2p (1-[(4-aminobenzoyl)indoline-5-sulfonyl])-4-phenyl-4,5-dihydroimidazolone) exhibits a cytotoxicity that is far more potent than doxorubicin and also exhibits highly effective antitumour activities against murine (3LL, Colon 26) and human xenograft (NCI-H23, SW620) tumor models.