Free-Standing Lipid Bilayers Based on Nanopore Array and Ion Channel Formation.

Integrated nanopores are novel and versatile single-molecule sensors for individual label-free biopolymer detection and characterization. However, their studies and application requires a stable lipid bilayer to maintain protein function. Herein, we describe a method for producing lipid bilayers across a nanopore array on a silicon nitride substrate. We used a painting technique commonly used with Teflon films to embed α-hemolysin (α-HL) into bilayer lipid membranes (BLMs) to form an ion channel. This was carried out in nanofluid developed in our lab. The membrane formation process, stability of BLMs and ion channel recordings were monitored by patch clamp in real-time. BLM formation was demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore in the range of ±100 mV. Membrane resistance (Rm) and capacitance (Cm) of the device with the bilayer were assessed by membrane test as above 1.0 GΩ and ~20±2 pF, respectively. The silicon nitride surface and aperture edge were smooth at the nanometer lever leading to remarkable membrane stability. The membrane lifetime was 5-24 h. A single α-HL channel inserted in 30-60 min applied a potential of +100 mV. The α-HL channel currents were recorded at ~100±10 pA. Such integrated nanopores enable analysis of channel functions under various solution conditions from the same BLM. This will open up a variety of applications for ion channels including high-throughput medical screening and diagnosis.

In vitro inhibition of tumor growth by low-dose iron oxide nanoparticles activating macrophages.

Macrophages as immunocyte are attracting more and more attention in cancer therapy. Our previous study observed that dimercaptosuccinic acid (DMSA)-coated Fe3O4 magnetic nanoparticles triggered comprehensive immune responses of mouse macrophages (RAW264.7 cells) and induced production of many kinds of cytokines. This study investigated the effects of Fe3O4 magnetic nanoparticles on RAW264.7 cells proliferation, migration, and inhibition of tumor growth in vitro. Fe3O4 magnetic nanoparticles had an average size of about 11 nm with good dispersibility and uniformity. Fe3O4 magnetic nanoparticles internalized efficiently into RAW264.7 cells. Through Cell Counting Kit-8 (CCK-8) detection, the proliferation of RAW264.7 cells significantly increased by the low-dose Fe3O4 magnetic nanoparticles (50 µg/mL) treatment. The results of wound-healing and Transwell assays both displayed a significant promotion of the RAW264.7 cells migratory capability compared with control group ( P<0.01). It is interesting to find that a large number of proliferated RAW264.7 cells were activated to surround quickly and attack mouse liver cancer cell (Hepa1-6) cells by Fe3O4 magnetic nanoparticles. The growth of Hepa1-6 cells was effectively inhibited according to microscope imaging and flow cytometry analysis. The inhibition may be cooperative effects of RAW264.7 cells proliferation, migration, and immune activation. The results suggest potential clinical value of low-dose iron oxide nanomaterials in cancer therapy.

Effect of Nb2O5 doping on improving the thermo-mechanical stability of sealing interfaces for solid oxide fuel cells.

Nb2O5 is added to a borosilicate sealing system to improve the thermo-mechanical stability of the sealing interface between the glass and Fe-Cr metallic interconnect (Crofer 22APU) in solid oxide fuel cells (SOFCs). The thermo-mechanical stability of the glass/metal interface is evaluated experimentally as well as by using a finite element analysis (FEA) method. The sealing glass doped with 4 mol.% Nb2O5 shows the best thermo-mechanical stability, and the sealing couple of Crofer 22APU/glass/GDC (Gd0.2Ce0.8O1.9) remains intact after 50 thermal cycles. In addition, all sealing couples show good joining after being held at 750 °C for 1000 h. Moreover, the possible mechanism on the thermo-mechanical stability of sealing interface is investigated in terms of stress-based and energy-based perspectives.

Detection of a single enzyme molecule based on a solid-state nanopore sensor.

The nanopore sensor as a high-throughput and low-cost technology can detect a single molecule in a solution. In the present study, relatively large silicon nitride (Si3N4) nanopores with diameters of ∼28 and ∼88 nm were fabricated successfully using a focused Ga ion beam. We have used solid-state nanopores with various sizes to detect the single horseradish peroxidase (HRP) molecule and for the first time analyzed single HRP molecular translocation events. In addition, a real-time monitored single enzyme molecular biochemical reaction and a translocation of the product of enzyme catalysis substrates were investigated by using a Si3N4 nanopore. Our nanopore system showed a high sensitivity in detecting single enzyme molecules and a real-time monitored single enzyme molecular biochemical reaction. This method could also be significant for studying gene expression or enzyme dynamics at the single-molecule level.

Single Nanoparticle Translocation Through Chemically Modified Solid Nanopore.

The nanopore sensor as a high-throughput and low-cost technology can detect single nanoparticle in solution. In the present study, the silicon nitride nanopores were fabricated by focused Ga ion beam (FIB), and the surface was functionalized with 3-aminopropyltriethoxysilane to change its surface charge density. The positively charged nanopore surface attracted negatively charged nanoparticles when they were in the vicinity of the nanopore. And, nanoparticle translocation speed was slowed down to obtain a clear and deterministic signal. Compared with previous studied small nanoparticles, the electrophoretic translocation of negatively charged polystyrene (PS) nanoparticles (diameter ~100 nm) was investigated in solution using the Coulter counter principle in which the time-dependent nanopore current was recorded as the nanoparticles were driven across the nanopore. A linear dependence was found between current drop and biased voltage. An exponentially decaying function (t d ~ e (-v/v0) ) was found between the duration time and biased voltage. The interaction between the amine-functionalized nanopore wall and PS microspheres was discussed while translating PS microspheres. We explored also translocations of PS microspheres through amine-functionalized solid-state nanopores by varying the solution pH (5.4, 7.0, and 10.0) with 0.02 M potassium chloride (KCl). Surface functionalization showed to provide a useful step to fine-tune the surface property, which can selectively transport molecules or particles. This approach is likely to be applied to gene sequencing.

DNA-functionalized silicon nitride nanopores for sequence-specific recognition of DNA biosensor.

Nanopores have been proven to be novel and versatile single-molecule sensors for individual unlabeled biopolymer detection and characterization. In the present study, a relatively large silicon nitride (Si3N4) nanopore with a diameter of approximately 60 nm was fabricated successfully using a focused Ga ion beam (FIB). We demonstrated a simple ex situ silanization procedure to control the size and functionality of solid-state nanopores. The presented results show that by varying the silanization time, it is possible to adjust the efficiency of probe molecule attachment, thus shrinking the pore to the chosen size, while introducing selective sensing probes. The functionalization of nanopores was verified by analysis of field-emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDS), and electrical measurements. Based on this study, we envision that the functionalized silicon nitride nanopores with the DNA probe might provide a biosensing platform for the detection and discrimination of a short single-stranded DNA oligomer of unknown sequences in the future.

A novel biosensor based on sol-gel poly () (PVA)/(titanium dioxide)TiO2 hybrid material.

A novel hydrogen peroxide biosensor has been fabricated based on Hb entrapped poly(vinyl alcohol) (PVA)/Titanium dioxide (TiO2) hybrid material. Multi-walled carbon nanotubes (MWCNTs) were then dispersed into the composite matrix. It was found that such hybrid material could retain the native biocatalytic activity of the entrapped Hb by electrochemical experiments. In addition, MWCNTs enhanced catalytic performance of hydrogen peroxide and promoted electronic transfer. Effects of some experimental variables such as the amount of MWCNTs, concentration of enzyme, amounts of modifier on the current response of the biosensor were investigated. A linear calibration graph was obtained in the concentration range of H2O2 from 0.5 to 2.7 µM (linear regression coefficient = 0.997) with a detection limit of 0.01 µM (S/N = 3). The apparent Michaelis-Menten constant Km was 0.997 µM. The biosensor displayed excellent repeatability, high sensitivity, long-term stability, and good selectivity. The recovery of H2O2 in samples was testified with satisfactory results.

Amperometric hydrogen peroxide biosensor based on immobilization of hemoglobin on a glassy carbon electrode modified with fe(3)o(4)/chitosan core-shell microspheres.

Novel magnetic Fe(3)O(4)/chitosan (CS) microspheres were prepared using magnetic Fe(3)O(4) nanoparticles and the natural macromolecule chitosan. Then, using an easy and effective hemoglobin (Hb) immobilization method, an innovative biosensor with a Fe(3)O(4)/CS-Hb-Fe(3)O(4)/CS "sandwich" configuration was constructed. This biosensor had a fast (less than 10 s) response to H(2)O(2) and excellent linear relationships were obtained in the concentration range of 5.0 × 10(-5) to 1.8 × 10(-3) M and 1.8 × 10(-3) to 6.8 × 10(-3) M with a detection limit of 4.0 × 10(-6) M (s/n = 3) under the optimum conditions. The apparent Michaelis-Menten constant K(m) was 0.29 mM and it showed the excellent biological activity of the fixed Hb. Moreover, the biosensor had long-time stability and good reproducibility. The method was used to determine H(2)O(2) concentration in real samples.