Silica gel solid nanocomposite electrolytes with interfacial conductivity promotion exceeding the bulk Li-ion conductivity of the ionic liquid electrolyte filler.

The transition to solid-state Li-ion batteries will enable progress toward energy densities of 1000 W·hour/liter and beyond. Composites of a mesoporous oxide matrix filled with nonvolatile ionic liquid electrolyte fillers have been explored as a solid electrolyte option. However, the simple confinement of electrolyte solutions inside nanometer-sized pores leads to lower ion conductivity as viscosity increases. Here, we demonstrate that the Li-ion conductivity of nanocomposites consisting of a mesoporous silica monolith with an ionic liquid electrolyte filler can be several times higher than that of the pure ionic liquid electrolyte through the introduction of an interfacial ice layer. Strong adsorption and ordering of the ionic liquid molecules render them immobile and solid-like as for the interfacial ice layer itself. The dipole over the adsorbate mesophase layer results in solvation of the Li+ ions for enhanced conduction. The demonstrated principle of ion conduction enhancement can be applied to different ion systems.

Optimization of the virus concentration method using polyethyleneimine-conjugated magnetic beads and its application to the detection of human hepatitis A, B and C viruses.

To enhance the sensitivity of virus detection by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads was developed in our previous study. However, several viruses could not be concentrated by this method. In this paper, the conditions of virus concentration were optimized to concentrate a wide range of viruses more efficiently. The PEI beads adsorbed viruses more efficiently than other cationic polymers, and the optimum virus concentration was obtained under weak acidic conditions. Mass spectrometric analysis revealed that several serum proteins, such as complement type 3, complement type 4 and immunoglobulin M (IgM), were co-adsorbed by the PEI beads, suggesting that the beads may adsorb viruses not only by direct adsorption, but also via immune complex formation. This hypothesis was confirmed by the result that poliovirus, which PEI beads could not adsorb directly, could be concentrated by the beads via immune complex formation. On the other hand, hepatitis A (HAV) and hepatitis C (HCV) viruses were adsorbed directly by PEI beads almost completely. Like poliovirus, hepatitis B virus (HBV) was concentrated efficiently by the addition of anti-HBV IgM. In conclusion, virus concentration using PEI beads is a useful method to concentrate a wide range of viruses and can be used to enhance the sensitivity of detection of HAV, HBV and HCV.

Anticoagulant mechanism of sulfonated polyisoprenes.

The influence of sulfonated polyisoprene (SPIP) on coagulation factors and human blood cells was investigated to elucidate and compare its anticoagulant mechanism with that of heparin. While the number of red cells was unaffected, the number of platelets decreased dramatically in the presence of SPIP due to aggregation. Using a synthetic peptide substrate to assay thrombin activity in the presence of its natural inhibitor, antithrombin (AT), we observed no stimulation by SPIP of AT-mediated inhibition. Nevertheless, thrombin cleavage of its natural substrate fibrinogen to fibrin peptide A was slightly inhibited. SPIP altered the electrophoretic mobility of fibrinogen and completely inhibited fibrinogen from clotting. We detected no significant influence of SPIP on factors II, VII, IX, and X, while factor XI and factors V and VIII were only slightly affected. Therefore, the main mechanism of SPIP's anticoagulant activity appears to be a strong interaction with fibrinogen and fibrin monomer, first, to prevent proteolytic conversion of the former to the latter and second, to inhibit polymerization of the fibrin monomer, once formed.

Virus concentration using polyethyleneimine-conjugated magnetic beads for improving the sensitivity of nucleic acid amplification tests.

To enhance the sensitivity of virus detection by polymerase chain reaction (PCR) and reverse-transcriptional (RT)-PCR, we developed a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads. PEI-magnetic beads adsorbed efficiently the enveloped viruses Sindbis virus and Herpes simplex 1 virus, and the nonenveloped virus SV-40, but not the nonenveloped viruses porcine parvovirus (PPV) or poliovirus, based on the PCR detection data. Furthermore, the infectivity in the supernatant of former viruses was reduced markedly after incubation with PEI-magnetic beads. Both real-time PCR and RT-PCR revealed that the DNA viruses were concentrated to a maximum of about 100 times the expected value, whereas the RNA viruses were concentrated over a thousand times, which was significantly more than expected. It was concluded that the PEI-magnetic beads are a superior novel means of concentrating viruses, with the exception of some non-enveloped viruses. The present method was found to enhance the sensitivity of virus detection by PCR and RT-PCR.

Virus concentration using sulfonated magnetic beads to improve sensitivity in nucleic acid amplification tests.

To enhance the sensitivity of virus detection by polymerase chain reaction (PCR) and reverse-transcriptional (RT)-PCR, we developed a novel virus-concentration method using sulfonated (SO-) magnetic beads in the presence of divalent cations. In the presence of either Zn(2+) or Cu(2+) ions, we showed that SO-magnetic beads were able to concentrate non-enveloped model viruses, such as porcine parvovirus (PPV) and poliovirus, which were not concentrated by polyethyleneimine (PEI)-magnetic beads.(1)) Using the SO-magnetic beads, the sensitivity of virus genome detection by PCR or RT-PCR can be enhanced. Therefore, an efficient virus concentration method using either SO-magnetic beads or PEI-magnetic beads enhances the sensitivity of virus detection by PCR or RT-PCR.

An improved method for detection of replication-competent retrovirus in retrovirus vector products.

Contamination by replication-competent retrovirus (RCR) is one of the most important safety issues of retrovirus vector products for gene therapy clinical research. To improve the sensitivity of RCR detection and to shorten the assay period, we have developed a novel RCR detection method (infectivity RT-PCR method) based on real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) in combination with virus infection and a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads. In this method, permissive cells were infected with RCR samples, and amplified RCR in the culture supernatants was adsorbed by PEI-beads. Then RCR RNA extracted from PEI-beads was quantified by real-time RT-PCR. We demonstrated that 1 infectious unit (iu) of RCR spiked in 10(6) cfu/ml of vector products could be detected within 3 days, and the sensitivity for viral detection was increased 3- to 10-fold compared with the direct S+L- assay. By this method, the presence of retroviral vector interfered with RCR detection only slightly. In conclusion, infectivity RT-PCR conducted in conjunction with virus concentration using PEI-beads can detect RCR more sensitively and rapidly than the conventional infectivity assay.

Detection of replication-competent adenoviruses spiked into recombinant adenovirus vector products by infectivity PCR.

The presence of replication-competent adenovirus (RCA) in clinical lots of adenovirus vectors raises a variety of safety concerns. To detect RCA in adenovirus vector products, the cell culture/cytopathic effect (CPE) method has generally been preferred. However, it is difficult to evaluate the amount of RCA clearly and quantitatively by this method. In addition, the cell culture/CPE method requires large-scale cell culturing and a substantial amount of time. For the purpose of establishing a method to detect RCA more sensitively and rapidly, we developed the infectivity PCR, a hybrid method that combines the infectivity assay and quantitative PCR. This method allows RCA to be quantified by real-time quantitative PCR using primers and a probe designed for E1 DNA. By infectivity PCR, 1 pfu of RCA spiked into 10(9) particles of adenovirus vectors could be detected. In contrast, CPE was observed in the cells infected with 10(4) pfu of RCA spiked into 10(9) particles of adenovirus vectors. The glass-beads method was suitable for extracting DNA rapidly from the RCA-infected cells. These results showed that infectivity PCR combined with the glass-beads-based DNA extraction method was useful for the detection of RCA in adenovirus vector products.

Development of real-time detection direct test for hepatitis B virus and comparison with two commercial tests using the WHO international standard.

AIMS: A highly reproducible and sensitive hepatitis B virus real-time detection direct (HBV RTD-direct) test using DNA extraction by magnetic beads coated with polyclonal anti-HBsAg, followed by the real-time detection polymerase chain reaction (PCR) method, was developed for the detection of HBV DNA. METHODS: The HBV DNA could be extracted from the HBsAg positive viral particles without resulting in viral DNA fragmentation. The HBV RTD-direct test was validated using a serial dilution panel of the WHO standard HBV DNA 97/746 I. RESULTS: The test had a dynamic range of 0.7-8.0 log10 international units (IU) per mL and the results were shown to be comparable to those obtained with two commercially available tests: the HBV DNA transcription-mediated amplification-hybridization protection assay and the Amplicor HBV Monitor test. In addition, the HBV RTD-direct test, based on magnetic extraction, successfully eliminated PCR inhibitors in clinical specimens. CONCLUSION: We conclude that the HBV RTD-direct test is an excellent alternative for monitoring patients undergoing antiviral treatment or for screening various clinical specimens.