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With a growing number of intracellular drug targets and the high efficacy of protein therapeutics, the targeted delivery of active proteins with negligible toxicity is a challenging issue in the field of precision medicine. Herein, a programed assembly of nucleoprotein nanoparticles (NNPs) using DNA and zinc fingers (ZnFs) for targeted protein delivery is presented. Two types of ZnFs with different sequence specificities are genetically fused to a targeting moiety and a protein cargo, respectively. Double-stranded DNA with multiple ZnF-binding sequences is grafted onto inorganic nanoparticles, followed by conjugation with the ZnF-fused proteins, generating the assembly of NNPs with a uniform size distribution and high stability. The approach enables controlled loading of a protein cargo on the NNPs, offering a high cytosolic delivery efficiency and target specificity. The utility and potential of the assembly as a versatile protein delivery vehicle is demonstrated based on their remarkable antitumor activity and target specificity with negligible toxicity in a xenograft mice model., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Molecular detection of target molecules with high sensitivity and specificity is of great significance in bio and medical sciences. Here, we present genetically functionalized ferritin nanoparticles with a high-affinity protein binder, and their utility as a signal generator in a variety of immunoassays and imaging. As a high-affinity protein binder, human IgG-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was used. The repebody was genetically fused to the N-terminal heavy-chain ferritin, and the resulting subunits were self-assembled to the repebody-ferritin nanoparticles composed of 24 subunits. The repebody-ferritin nanoparticles were shown to have a three-order of magnitude higher binding affinity toward human IgG than free repebody mainly owing to a decreased dissociation rate constant. The repebody-ferritin nanoparticles were conjugated with fluorescent dyes, and the resulting nanoparticles were used for western blotting, cell imaging, and flow cytometric analysis. The dye-labeled repebody-ferritin nanoparticles were shown to generate about 3-fold stronger fluorescent signals in immunoassays than monovalent repebody. The repebody-functionalized ferritin nanoparticles can be effectively used for sensitive and specific immunoassays and imaging in many areas., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The integration of a targeted delivery with a tumour-selective agent has been considered an ideal platform for achieving high therapeutic efficacy and negligible side effects in cancer therapy. Here, we present engineered protein nanoparticles comprising a tumour-selective oncolytic protein and a targeting moiety as a new format for the targeted cancer therapy. Apoptin from chicken anaemia virus (CAV) was used as a tumour-selective apoptotic protein. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumour-targeting moiety and a fusion partner for producing apoptin nanoparticles in E. coli, respectively. The repebody was genetically fused to apoptin, and the resulting fusion protein was shown to self-assemble into supramolecular repebody-apoptin nanoparticles with high homogeneity and stability as a soluble form when expressed in E. coli. The repebody-apoptin nanoparticles showed a remarkable anti-tumour activity with negligible side effects in xenograft mice through a cooperative action of the two protein components with distinct functional roles. The repebody-apoptin nanoparticles can be developed as a systemic injectable and tumour-selective therapeutic protein for targeted cancer treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We describe a chip-based method to detect protein glycosylation based on the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs). Our assay system relies on modulations in the energy transfer between the nanoparticles on a surface. The photoluminescence (PL) of lectin-coated QDs (energy donor) immobilized on a glass slide is quenched by carbohydrate-coated AuNPs (energy acceptor), and the presence of the glycoprotein causes the increase of the PL of QDs. As a proof-of-concept, Concanavalin A-coated QDs (ConA-QDs) and dextran-coated AuNPs (Dex-AuNPs) were used to detect the mannosylated proteins. As a result, the PL intensity of QDs was found to be linearly correlated with the concentration and the number of glycan moiety of the glycoprotein. We anticipated that our simple assay system will find applications for the analysis of glycoproteins with high selectivity and sensitivity in a high-throughput manner.

We demonstrate that protein kinase can be assayed with high sensitivity on peptide-conjugated gold nanoparticles (AuNPs). Phosphorylation of peptides on the AuNP-monolayers was detected by using an anti-phosphotyrosine antibody (alpha-pY) and Cy3-labeled secondary antibody (Cy3-alpha-mIgG) as a probing molecule. When compared to conventional self-assembled monolayers (SAMs), spherical and three-dimensional geometry of AuNPs led to high surface density of peptide substrate and easy accessibility to enzyme, and consequently the resulting AuNP monolayers gave rise to improved detection sensitivity. Blocking of peptide-conjugated AuNPs with a poly(ethylene glycol) (PEG) also contributed to a higher signal-to-background ratio in kinase and its inhibition assays. The use of AuNPs as the platform surface will enable highly sensitive detection of protein kinases in a high-throughput manner.

We demonstrate that magnetic mesocellular carbon foams (Mag-MCF-C) can be effectively used for enrichment and desalting of protein digests or peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The large mesocellular pores and surface area of Mag-MCF-C are likely to mainly contribute to high efficiency in enrichment and desalting of protein digests. The magnetic property of Mag-MCF-C enabled easy and simple enrichment and desalting process comprising adsorption, washing, and separation steps by using an external magnet. Following elution from Mag-MCF-C by using a matrix solution (CHCA in 70% ACN/0.1% TFA), the peptides were subjected to MALDI-MS analysis. As a result, MALDI mass spectra of peptides or tryptic protein digests were distinct even at a peptide concentration as low as 50 pM. The use of Mag-MCF-C resulted in significantly improved sequence coverage for protein identification when compared to other conventional methods. Mag-MCF-C will find applications in mass spectrometric analysis of low abundance peptides or protein digests with high sensitivity., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

We demonstrate the use of gold nanoparticles (AuNPs) to enhance the secondary ion emission of peptides in time-of-flight secondary ion mass spectrometry (TOF-SIMS). The signal intensity of peptides adsorbed onto AuNPs was significantly increased when compared to that of self-assembled monolayers (SAMs). This gold nanoparticle-enhanced SIMS, termed NE-SIMS, enabled the sensitive detection of subtle modifications of peptides, such as phosphorylation. From a quantitative analysis of the amounts of adsorbed peptides and AuNPs on SAMs using quartz crystal microbalance and surface plasmon resonance spectroscopy, the ratio of peptide molecule to AuNP on amine-SAMs was revealed to be 18-19:1. When considering the ratio of peptide to matrix (1:10(3)-10(6)) employed in a matrix-enhanced SIMS, the use of AuNPs gave rise to a significantly increased secondary ion emission of peptides. Peptides were adsorbed onto patterned AuNPs on SAMs using a microfluidic system, and well-contrasted molecular ion images were obtained. NE-SIMS is expected to be applied to a chip-based analysis of modification of biomolecules in a label-free manner.

Nanoparticle clusters (NPCs) have attracted significant interest owing to their unique characteristics arising from their collective individual properties. Nonetheless, the construction of NPCs in a structurally well-defined and size-controllable manner remains a challenge. Here we demonstrate a strategy to construct size-controlled NPCs using the DNA-binding zinc finger (ZnF) protein. Biotinylated ZnF was conjugated to DNA templates with different lengths, followed by incubation with neutravidin-conjugated nanoparticles. The sequence specificity of ZnF and programmable DNA templates enabled a size-controlled construction of NPCs, resulting in a homogeneous size distribution. We demonstrated the utility of magnetic NPCs by showing a three-fold increase in the spin-spin relaxivity in MRI compared with Feridex. Furthermore, folate-conjugated magnetic NPCs exhibited a specific targeting ability for HeLa cells. The present approach can be applicable to other nanoparticles, finding wide applications in many areas such as disease diagnosis, imaging, and delivery of drugs and genes. [ABSTRACT FROM AUTHOR]

The main contribution of this paper is the development of a novel process for the formation of copper indium gallium diselenide (CIGS) films. CIGS films with a thickness of 4 μm and grain size from 0.3 μm to 1 μm were prepared from a Cu(InGa) (CIG) metallic alloy and Se nanoparticles by the intense pulsed light (IPL) technique. The melting of the CIG and Se nanoparticles and nucleation of CIGS occurred in a very short reaction time of 2 ms. It is believed that the Se diffuses into the CIG lattice to form the CIGS chalcopyrite crystal structure. The tetragonal chalcopyrite crystal structure was confirmed by x-ray powder diffraction (XRD), while the microstructure and composition were determined by field-emission scanning electron microscopy (FESEM), energy-dispersive x-ray spectroscopy (EDAX), and x-ray fluorescence (XRF) spectroscopy. [ABSTRACT FROM AUTHOR]

Most commercial copper nanoparticles are covered with an oxide shell and cannot be sintered into conducting lines/films by conventional thermal sintering. To address this issue, past efforts have utilized complex reduction schemes and sophisticated chambers to prevent oxidation, thereby rendering the process cost ineffective. To alleviate these problems, we demonstrate a reactive sintering process using intense pulsed light (IPL) in the present study. The IPL process successfully removed the oxide shells of copper nanoparticles, leaving a conductive, pure copper film in a short period of time (2 ms) under ambient conditions. The in situ copper oxide reduction mechanism was studied using several different experiments and analyses. We observed instant copper oxide reduction and sintering through poly( N-vinylpyrrolidone) functionalization of copper nanoparticles, followed by IPL irradiation. This phenomenon may be explained by oxide reduction either via an intermediate acid created by ultraviolet (UV) light irradiation or by hydroxyl (-OH) end groups, which act like long-chain alcohol reductants. [ABSTRACT FROM AUTHOR]

In this work, a flash-light sintering process for Cu nanoinks was studied. In order to precisely monitor the milliseconds flash-light sintering process, a real-time Wheatstone bridge electrical circuit and a high-rate data acquisition system were used. The effects of several flash-light irradiation conditions (irradiation energy, pulse number, on-time, and off-time) and the effects of the amount of poly(N-vinylpyrrolidone) in the Cu nanoink on the flash-light sintering process were investigated. The microstructures of the sintered Cu films were analyzed by scanning electron microscopy. To investigate the oxidation or reduction of the oxide-covered copper nanoparticles, a crystal phase analysis using x-ray diffraction was performed. In addition, the sheet resistance of Cu film was measured using a four-point probe method. From this study, it was found that the flash-light sintered Cu nanoink films have a conductivity of 72 Ωm/sq without any damage to the polyimide substrate. Similar nanoinks are expected to be widely used in printed and flexible electronics products in the near future. [ABSTRACT FROM AUTHOR]

In this work, the hybrid copper inks with precursor and nanoparticles were fabricated and sintered via flash light irradiation to achieve highly conductive electrode pattern with low porosity. The hybrid copper ink was made of copper nanoparticles and various copper precursors (e.g., copper(II) chloride, copper(II) nitrate trihydrate, copper(II) sulfate pentahydrate and copper(II) trifluoroacetylacetonate). The printed hybrid copper inks were sintered at room temperature and under ambient conditions using an in-house flash light sintering system. The effects of copper precursor weight fraction and the flash light irradiation conditions (light energy and pulse duration) were investigated. Surfaces of the sintered hybrid copper patterns were analyzed using a scanning electron microscope. Also, spectroscopic characterization techniques such as Fourier transform infrared spectroscopy and X-ray diffraction were used to investigate the crystal phases of the flash light sintered copper precursors. High conductivity hybrid copper patterns (27.3 μΩ cm), which is comparable to the resistivity of bulk copper (1.68 μΩ cm) were obtained through flash light sintering at room temperature and under ambient conditions. [ABSTRACT FROM AUTHOR]

Graphical abstract Scalable Flash White Light (FWL) sintering of copper nanoparticles on silicon wafer is used to create highly conductive electrodes for crystalline silicon solar cells. After 1 s of FWL sintering, the Cu electrode has high electrical conductivity with lower cost, and no thermal damage, as compared to the state-of-the-art. Highlights • A new way to fabricate Cu electrodes using flash white light is demonstrated. • Flash white light sintering process is dramatically simple and high speed process. • Multi-pulsed FWL sintering method was developed for crystalline Si solar cells. • Artificial oxidation treatment improves the FWL sintering of Cu NPs on Si wafer. • Highly conductive Cu films were successfully produced under ambient conditions. Abstract In this work, ultra-high speed flash white light (FWL) sintering method of copper nanoparticle pastes on silicon wafer substrate, was developed to produce highly conductive and low-cost copper electrodes for crystalline silicon solar cells. FWL sintering of copper nanoparticles on silicon wafer substrate has been regarded to be very difficult, due to its high thermal conductivity (k) compared with that of polymer (PI and PET) substrates. To overcome this limitation, we applied multiple pulsed FWL to sinter copper nanoparticles (Cu NPs) printed on silicon wafer. Furthermore, bimodal Cu NPs with different size were also applied to enhance the packing density of Cu films for highly conductive Cu electrodes. Finally, this work demonstrated that Cu NP-pastes are successfully sintered on crystalline silicon wafer substrate by multiple pulsed FWL irradiations. [ABSTRACT FROM AUTHOR]

• The Mo pattern was printed by screen printing method using conductive ink on Si wafer. • The printed Mo pattern was successfully sintered by intense pulsed light process (35 μΩ·cm). • The thin film transistor was realized using Mo pattern as source and drain electrode. • The noteworthy saturation mobility of thin film transistor was 13.5 cm2·(V·s)−1. In this study, the molybdenum (Mo) pattern was realized via intense pulsed light (IPL) irradiation process under ambient condition, without vacuum deposition process. The screen printing method was applied to realize a certain pattern using conductive ink composed of Mo nanoparticles. This printed Mo pattern showed low electrical conductivity due to oxide film on the nanoparticles surface. To improve this drawback, the IPL irradiation process was optimized for sintering of Mo pattern, therefore the remarkable electrical conductivity (35 μ Ω ·cm) was observed after IPL irradiation process. To investigate the microstructure and other reaction effects of Mo pattern, the various analysis was conducted such as scanning electron microscope, X-ray diffraction, focused ion beam analysis. This printed Mo pattern was applied for the source and drain (S/D) electrode of indium gallium zinc oxide (IGZO) based thin film transistor (bottom gate; TFT). The interfacial microstructure between Mo pattern and IGZO layer was investigated by transmittance electron microscope analysis. Consequently, the TFT with IPL-sintered Mo (S/D) pattern showed a noteworthy saturation mobility of 13.5 cm2·(V·s)−1. [ABSTRACT FROM AUTHOR]

The effect of poly (N-vinylpyrrolidone) (PVP) molecular weight (MW) on the flash light sintering of copper nanopaste was investigated. The copper nanopaste was coated on polyimide substrates using doctor blade method. PVP was used as reducing agent to functionalize the copper nanoparticles during flash light sintering process. To find the optimum sintering conditions of copper nanoparticles, the various MWs of PVP (10 000, 40 000 and 55 000) were used and the flash light irradiation energy was varied from 7.5 J/cm 2 to 17.5 J/cm 2 . Meanwhile, other flash light irradiation conditions (pulse numbers, on-time and off-time durations) and amounts of PVP were fixed to clarify the effect of the PVP MW. As the results, it was found that flash light sintered copper nanofilms have the resistivity of 54 μΩcm without any damages to the polymer substrate. To characterize the microstructures and transformation crystal phase of the sintered copper nanofilms, scanning electron microscopy and X-ray diffraction were conducted. In order to understand the interaction between oxidized copper and PVP, X-ray photoelectron spectroscopy was analyzed. To observe the electrical conductivity of flash light sintered copper nanofilms, the sheet resistance of the sintered copper nanofilms was measured using a four-point probe method and the sheet resistance changes during the flash light irradiation process were monitored using in-situ monitoring system. [ABSTRACT FROM AUTHOR]

Abstract: We synthesized Pt, Pt–Ru alloy and Pt–Ru–Mo alloy nanoparticles on the multi-walled carbon nanotubes (MWCNTs) using flash light irradiation and characterized these catalysts. Pt100, Pt50-Ru50 alloy and Pt43-Ru43-Mo14 alloy are coated onto MWCNTs followed by flash light irradiation to facilitate the formation of nanoparticle-alloys. The fabricated pure Pt and Pt-based alloy nanoparticle/MWCNTs were characterized by X-ray diffraction (XRD), raman spectroscopy and scanning electron microscopy (SEM). Cyclic voltammetry (CV) studies and electrochemical impedance spectroscopy (EIS) results revealed that the Pt43-Ru43-Mo14/MWCNT catalyst has higher activity and stability with regard to methanol electro-oxidation than the Pt100/MWCNT and Pt50-Ru50/MWCNT catalysts. [Copyright &y& Elsevier]

Abstract: We demonstrate to decorate carbon nanotubes (CNTs) with metal alloy nanoparticles by flash light from a xenon lamp highlighting dramatically simplified process and ultrafast speed of millisecond. By managing the pulse energy of the irradiation, we customize the nucleation and growth rates of homogeneous binary and ternary nanoparticles with Pt–Ru and Pt–Ru–Mo thin layers that are e-beam deposited onto CNTs. No damage of CNTs during the light-induced nanogranulation is guaranteed by the rapid and surface-localized heating mechanism. The fabricated nanoparticle/CNT nanostructures are characterized using variety of microscopic and spectroscopic techniques such as SEM, TEM and Raman spectroscopy. [Copyright &y& Elsevier]

Abstract: This paper demonstrates a new magnetophoretic position detection method for multiplexed immunoassay using colored microspheres as an encoding tool in a microchannel. Colored microspheres conjugated with respective capture molecules are incubated with a mixture of target analytes, followed by reaction with the probe molecules which had been conjugated with superparamagnetic nanoparticles (SMNPs). Under the magnetic field gradient, the resulting microspheres are deflected from their focused streamlines in a microchannel, and respective colored microspheres are detected using color charge-coupled device (CCD) in a specific detection region of the microchannel. The color and position of respective colored microspheres are automatically decoded and analyzed by MATLAB program, and the position was correlated with the concentration of corresponding target analytes. As a proof-of-concept, we attempted to assay simultaneously three types of biotinylated immunoglobuline Gs (IgGs), such as goat, rabbit and mouse IgGs, using colored microspheres (red, yellow and blue, respectively). As the capture molecules, corresponding anti-IgGs were employed and target analytes were probed using streptavidin-modified superparamagnetic nanoparticles. As a result, three analytes were simultaneously assayed using colored microspheres with high accuracy, and detection limits of goat IgG, rabbit IgG and mouse IgG were estimated to be 10.9, 30.6 and 12.1fM, respectively. In addition, with adjustment of the flow rate and detection zone, the dynamic range could be controlled by more than one order of magnitude. [Copyright &y& Elsevier]

Abstract: Enhancement of signals in time-of-flight secondary ion mass spectrometry (ToF-SIMS) studies is necessary to many biological applications. We have developed an efficient method of enhancing the signals of secondary ions from peptides using gold nanoparticles (AuNPs) attached to a well-controlled surface such as self-assembled monolayers (SAMs). AuNPs function as both signal enhancers and effective binding sites for peptides, which allow the high signal intensity from the peptides to produce well-contrasted ToF-SIMS images of peptides that are micropatterned on the surface of the AuNPs. For application, this AuNP-enhanced SIMS (NE-SIMS) provided the basis for the spectrum and images to assay protein kinases and their inhibitors. Phosphorylation efficiency and inhibitor effect were quantified by detecting mass change of the peptide substrates by kinase reaction. Maximum efficiency of phosphorylation was achieved from cysteine-tethered peptides with a spacer linker, indicating that accessibility of kinase was dependent on the surface orientation and length of the peptide substrate on the three-dimensional AuNPs. The activities of other enzymes such as phosphatase and protease could also be assayed using NE-SIMS. Our study shows that NE-SIMS can be applied as a useful tool for enzyme assay in biochip surfaces. [Copyright &y& Elsevier]