Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay.

BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. METHODS: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays. RESULTS: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3' termini of each genomic segment and subsequent qPCR of the 5' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R2 = 0.931, P = 0.000066). CONCLUSIONS: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.

Sensitive Detection of C-Reactive Protein by One-Step Method Based on a Waveguide-Mode Sensor.

One-step biosensing methods enable the quick and simplified detection of biological substances. In this study, we developed a sensitive one-step method on the basis of a waveguide-mode sensor, which is an optical sensor utilizing waveguide-mode resonance and evanescent light. Streptavidin-conjugated and gold-nanoparticle-conjugated antibodies were reacted with a target substance and applied onto a biotinylated sensing plate. The target substance was detected by observing changes in sensor signals caused by binding the immunocomplex to the sensing surface. Performance of the developed one-step method was examined using a C-reactive protein (CRP) as a target substance. A sensor signal corresponding to the concentration of CRP was obtained. The minimal detectable CRP concentration of the developed method was 10 pM. The developed method greatly simplifies quantitative protein detection without reducing sensitivity.

Application of a Waveguide-Mode Sensor to Blood Testing for Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus and Treponema pallidum Infection.

Testing for blood-transmitted infectious agents is an important aspect of safe medical treatment. During emergencies, such as significant earthquakes, many patients need surgical treatment and/or blood transfusion. Because a waveguide mode (WM) sensor can be used as a portable, on-site blood testing device in emergency settings, we have previously developed WM sensors for detection of antibodies against hepatitis B virus and hepatitis C virus and for forward ABO and Rh(D) and reverse ABO blood typing. In this study, we compared signal enhancement methods using secondary antibodies conjugated with peroxidase, a fluorescent dye, and gold nanoparticles, and found that the peroxidase reaction method offers superior sensitivity while gold nanoparticles provide the most rapid detection of anti-HBs antibody. Next, we examined whether we could apply a WM sensor with signal enhancement with peroxidase or gold nanoparticles to detection of antibodies against hepatitis C virus, human immunodeficiency virus and Treponema pallidum, and HBs antigen in plasma. We showed that a WM sensor can detect significant signals of these infectious agents within 30 min. Therefore, a portable device utilizing a WM sensor can be used for on-site blood testing of infectious agents in emergency settings.

Development of a TiO2/SiO2 waveguide-mode chip for an ultraviolet near-field fluorescence sensor.

Aimed at detecting fluorescent-labeled biological substances sensitively, a sensor that utilizes near-field light has attracted much attention. According to our calculations, a planar structure composed of two dielectric layers can enhance the electric field of UV near-field light effectively by inducing waveguide-mode (WM) resonance. The fluorescence intensity obtainable by a WM chip with an optimized structure is 5.5 times that obtainable by an optimized surface plasmon resonance chip. We confirmed the above by making a WM chip consisting of TiO2 and SiO2 layers on a silica glass substrate and by measuring the fluorescence intensity of a solution of quantum dots dropped on the chip.

Optimization of a waveguide-mode sensing chip for an ultraviolet near-field illumination biosensor.

A waveguide-mode sensor with a planar sensing chip, consisting of two waveguiding layers and a glass substrate, is a promising candidate for a near-field illumination biosensor. Aiming at using fluorescent labeling induced by ultraviolet light, we optimize the structure of a waveguide-mode sensing chip, based on the mechanism for enhancing ultraviolet near-field light revealed by numerical calculations. Candidates of optimal materials are also presented. The chip optimized as above should be able to enhance the intensity of ultraviolet near-field light 25 times as high as an Al surface plasmon resonance sensing chip.

Carbon Nanotubes as Fluorescent Labels for Surface Plasmon Resonance-Assisted Fluoroimmunoassay.

The photoluminescence properties of carbon nanotubes (CNTs), including the large Stokes shift and the absence of fluorescent photobleaching, can be used as a fluorescent label in biological measurements. In this study, the performance of CNTs as a fluorescent label for surface plasmon resonance (SPR)-assisted fluoroimmunoassay is evaluated. The fluorescence of (8, 3) CNTs with an excitation wavelength of 670 nm and an emission wavelength of 970 nm is observed using a sensor chip equipped with a prism-integrated microfluidic channel to excite the SPR. The minimum detectable concentration of a CNT dispersed in water using a visible camera is 0.25 µg/mL, which is equivalent to 2 × 1010 tubes/mL. The target analyte detection using the CNT fluorescent labels is theoretically investigated by evaluating the detectable number of CNTs in a detection volume. Assuming detection of virus particles which are bound with 100 CNT labels, the minimum number of detectable virus particles is calculated to be 900. The result indicates that CNTs are effective fluorescent labels for SPR-assisted fluoroimmunoassay.

Parallel-incidence-type waveguide-mode sensor with spectral-readout setup.

A waveguide-mode sensor of the spectral-readout type can be used to detect changes in the complex refractive index in the vicinity of the surface of a sensing plate by observing the change in the spectrum of light reflected on the surface. The sensor's configuration can be simplified by adopting a parallel-incidence-type optical setup. To obtain a high sensitivity, the optimization of the sensing-plate structure, incidence angle, and detection wavelength band is essential for the sensor. In the present report, the results predicted by simulations are compared with experimental results in order to evaluate their validity. A discussion of the optimal design for the parallel-incidence-type sensor is also presented, according to the results obtained.

Generation of anti-influenza aptamers using the systematic evolution of ligands by exponential enrichment for sensing applications.

The systematic evolution of ligands by exponential enrichment (SELEX) is a selection process for identifying high-affinity selective molecules from a randomized combinatorial nucleic acid library against a wide range of target molecules. Using a pool of N25 RNA molecules, the SELEX process was performed against two targets from influenza viruses, namely, intact influenza B/Tokio/53/99 and hemagglutinin of infuluenza B Jilin/20/2003. The selection processes were evaluated by surface plasmon fluorescence spectroscopy (SPFS), and the result was compared to that obtained by a conventional radioisotope method. Clear discrimination among different selection cycles was displayed by SPFS, indicating that this method can be used as an alternative method of radioisotope labeling. The dissociation constant of the selected aptamers against the targets was in the low nanomolar range. The sensitivity of the selected aptamer against intact influenza B/Tokio/53/99 to detect the influenza virus was the low ng/mL level, an approximately 250-fold higher sensitivity than that of the commercially obtained antibody. The target binding sites on the aptamer were predicted by mapping analyses. The selected aptamer could discriminate other influenza strains, and the sensitivity of the selected aptamer was further confirmed by gold-nanoparticle-based sensing on a waveguide-mode sensor. This finding demonstrates that the selected aptamer would be useful for detecting influenza viruses at an early stage of infection and for the purpose of influenza surveillance.

Design and fabrication of biosensing interface for waveguide-mode sensor.

In order to develop a biosensing system with waveguide-mode sensor, fabrication of a biosensing interface on the silica surface of the sensing chip was carried out using triethoxysilane derivatives with anti-leptin antibody. Triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties were synthesized to immobilize the antibody and to suppress nonspecific adsorption of proteins, respectively. The chip modified with triethoxysilane derivatives bearing oligoethylene glycol moiety suppressed nonspecific adsorption of proteins derived from human serum effectively by rinse with PBS containing surfactant (0.05% Tween 20). On the other hand, it was confirmed that antibody was immobilized on the chip by immersion into antibody solution to show response of antigen-antibody reaction, where the chip was modified with triethoxysilane derivatives bearing succinimide ester moiety. When the interface was fabricated with antibody and a mixture of triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties, the response of antigen-antibody reaction depended on composition of the mixture and enhanced with the increase of ratio for triethoxysilane derivatives bearing succinimide ester moiety reflecting the antibody concentration immobilized on the chip. While introduction of excess triethoxysilane derivatives bearing succinimide ester moiety induced nonspecific adsorption of proteins derived from human serum, the immobilized antibody on the chip kept its activity after 1-month storage in a refrigerator. Taking into consideration those factors, the biosensing interface was fabricated using triethoxysilane derivatives with anti-leptin antibody to examine performance of the waveguide-mode sensor. It was found that the detection limits for human leptin were 50 ng/mL in PBS and 100 ng/mL in human serum. The results demonstrate that the waveguide-mode sensor powered by the biosensing interface fabricated with those triethoxysilane derivatives and antibody has potential to detect several tens of nanograms per milliliter of biomarkers in human serum with an unlabeled detection method.

A high-performance waveguide-mode biosensor for detection of factor IX using PEG-based blocking agents to suppress non-specific binding and improve sensitivity.

An evanescent-field-coupled waveguide-mode (EFC-WM) sensor utilizes monolithic SiO2/Si/SiO2 sensing plates having a multilayered structure and is used to evaluate a blocking agent comprising poly(ethylene glycol)-based block copolymers. Factor IX (FIX) protein was detected using its aptamer, viz. FIX was immobilized on a glutaraldehyde-modified silica surface, and then treated with a biotinylated aptamer. The quantitative analysis of FIX was carried out using streptavidin-conjugated gold nanoparticles (SA-GNPs). The blocking polymer, poly(ethylene glycol)-b-poly(acrylic acid) (PEG-b-PAAc), was found to mask unreacted amine and glutaraldehyde (Glu) moieties on the SiO2 surface, and it completely prevented the non-specific binding of SA-GNPs. By exploiting the strong blocking effect of PEG-b-PAAc, we achieved high ligand-analyte interaction sensitivity (sensitive down to 100 pM). To improve the sensitivity further, we also used pentaethylenehexamine-terminated PEG (N6-PEG) on GNPs. The improvement in sensitivity was found to be 1000-fold (to 100 fM), which was substantiated by the observation of higher numbers of GNPs on the sensing surface in the results of the scanning electron microscopic examination. Based on the competition assay of free biotin premixed with SA-GNPs, it was concluded that some active biotin-binding sites on the streptavidin were blocked by N6-PEG, which improved the binding ability to the biotinylated sensing surface. An optimum number of binding sites on the SA-GNPs might improve their binding affinity. The strategy shown with dual polymers, viz. blocking of the sensor chip surface and coating of SA-GNPs, is recommended for developing sensors with higher sensitivity and reliability. Selective binding of the aptamer to a very small amount of FIX in the mixed sample containing FXIa and FVIIa, or albumin, makes this the optimal strategy for detecting a FIX deficiency in human blood samples.

Surface functionalization chemistries on highly sensitive silica-based sensor chips.

The surfaces of silica-based sensor chips, designed for evanescent-field-coupled waveguide-mode sensors, were functionalized using various surface chemistries. The immobilization of molecular entities on the functionalized silica surfaces was monitored using various microscopic techniques (scanning electron, fluorescence, and atomic force microscopies). Further, gold nanoparticle-based signal enhancement analyses were performed with protein conjugation on different functionalized surfaces using a waveguide-mode sensor. Based on these analyses, the sensor surfaces modified with glutaraldehyde (Glu) and carbonyldiimidazole were found to be good for molecules of different sizes. In addition, it can be inferred that the Glu-modified surface may be suitable for small molecules with diameters around 5 nm owing to its surface roughness. The modified surface with carbonyldiimidazole is suitable for the direct immobilization of larger molecules especially for biomolecular assemblies without intermediate chemical modifications.

Waveguide-mode sensors as aptasensors.

Aptamers are artificial nucleic acid ligands that can be generated by in vitro selection through partition and amplification. Aptamers can be generated against a wide range of biomolecules through the formation of versatile stem-loop structures. Because aptamers are potential substitutes for antibodies and drugs, the development of an aptamer-based sensor (aptasensor) is mandatory for diagnosis. We previously reported that waveguide-mode sensors are useful in the analysis of a wide range of biomolecular interactions, including aptamers. The advantages of the waveguide-mode sensor that we developed include physical and chemical stability and that higher sensitivity can be achieved with ease by perforating the waveguide layer or using colored materials such as dyes or metal nanoparticles as labels. Herein, we provide an overview of the strategies and applications for aptamer-based analyses using waveguide-mode sensors.

Quick and ultra-sensitive digital assay of influenza virus using sub-picoliter microwells.

Quick and sensitive virus detection methods have been developed using sub-picoliter microwells. One method uses an aggregation-induced emission (AIE) reagent, and the other uses an enzymatic reaction supported with immunomagnetic beads at a high concentration of 108 particles/mL. Examination of influenza A virus detection using the AIE reagent exhibited a detection limit of 3 × 105 copies/mL, which was achievable within 1 min of the total measurement time. The high-concentration immunomagnetic beads method exhibited a detection limit as low as 1 × 102 copies/mL. The developed methods are effective and practical tools for ultrafast and ultrasensitive virus detection.

Optimal design of a spectral readout type planar waveguide-mode sensor with a monolithic structure.

Optical planar waveguide-mode sensor is a promising candidate for highly sensitive biosensing techniques in fields such as protein adsorption, receptor-ligand interaction and surface bacteria adhesion. To make the waveguide-mode sensor system more realistic, a spectral readout type waveguide sensor is proposed to take advantage of its high speed, compactness and low cost. Based on our previously proposed monolithic waveguide-mode sensor composed of a SiO2 waveguide layer and a single crystalline Si layer [1], the mechanism for achieving high sensitivity is revealed by numerical simulations. The optimal achievable sensitivities for a series of waveguide structures are summarized in a contour map, and they are found to be better than those of previously reported angle-scan type waveguide sensors.

Shape-sensitive reflectance by nanostructured metal attached on an optical waveguide-mode sensor.

The optical reflectance of He-Ne laser light on a waveguide-mode sensor was measured as a function of light incident angle, in the case of either a metal (Au, Cr or Pt) film or nanoparticles being attached to the waveguide surface of the sensor. A dip appears in the reflectance spectrum as a function of incident angle at the angle where waveguide-mode excitation is induced. It is found that the dip moves toward a lower angle in the case that the attached metal is of a film shape, while it shifts toward a higher angle when the metal is an ensemble of nanoparticles. This difference in the direction of shift can be explained well by theoretical calculations using average refractive indices of the metal-containing layers. The present result indicates that one can estimate whether a metal nanostructure is film-like or an ensemble of spherical nanoparticles by the sensor.

Detection of colored nanomaterials using evanescent field-based waveguide sensors.

We have developed an optical system designed for detecting colored nanomaterials in aqueous solutions, using the concept of evanescent-field-coupled waveguide-mode sensors. In this study, we found that the waveguide modes induced in the sensor are intrinsically sensitive to a change in optical absorption, or a 'change in color'. The system detects less than one gold nanoparticle (diameter: 20 nm) adsorbed per square micrometer. It is also demonstrated that significant signal enhancement due to adsorption of molecules is achieved using a dye. The developed sensor rarely suffers from a drawback of impurity adsorption. The system is expected to be applied as an effective sensing tool for metal colloids, nanoparticles, and colored biomolecules in solution.

Evaluation of nucleic acid duplex formation on gold over layers in biosensor fabricated using Czochralski-grown single-crystal silicon substrate.

With a view to developing an economical and elegant biosensor chip, we compared the efficiencies of biosensors that use gold-coated single-crystal silicon and amorphous glass substrates. The reflectivity of light over a wide range of wavelengths was higher from gold layer coated single-crystal silicon substrates than from glass substrates. Furthermore, the efficiency of reflection from gold layers of two different thicknesses was examined. The thicker gold layer (100 nm) on the single-crystal silicon showed a higher reflectivity than the thinner gold film (10 nm). The formation of a nucleic acid duplex and aptamer-ligand interactions were evaluated on these gold layers, and a crystalline silicon substrate coated with the 100-nm-thick gold layer is proposed as an alternative substrate for studies of interactions of biomolecules.

Plasmonic activity on gold nanoparticles embedded in nanopores formed in a surface layer of silica glass by swift-heavy-ion irradiation.

Silica glass was irradiated by swift heavy ions by selecting the ion species and its energy in order to induce the largest damaged regions. These regions were then selectively etched by hydrofluoric acid vapour to form nanopores on the glass surface. Subsequently, gold nanoparticles were embedded into the nanopores by vacuum evaporation, followed by thermal treatment. In the new plasmonic structure obtained with these procedures, the localized surface plasmon excitation wavelength induced around the gold nanoparticles was found to show a redshift, which agreed well with the theoretical calculation, when water was introduced into the nanopores. This indicates that the fabricated structure can be used as a sensing element to detect the adhesion of substances such as biomolecules to the nanoparticles by measuring the redshift.

Monitoring surface-assisted biomolecular assembly by means of evanescent-field-coupled waveguide-mode nanobiosensors.

Biological self-assembly is a natural process that involves various biomolecules, and finding the missing partner in these interactions is crucial for a specific biological function. Previously, we showed that evanescent-field-coupled waveguide-mode sensor in conjunction with a SiO(2) waveguide, the surfaces which contain cylindrical nanometric holes produced by atomic bombardment, allowed us to detect efficiently the biomolecular interactions. In the present studies, we showed that the assembly of biomolecules can be monitored using the evanescent-field-coupled waveguide-mode biosensor and thus provide a methodology in monitoring assembly process in macromolecular machines while they are assembling.

A plasmonic photocatalyst consisting of silver nanoparticles embedded in titanium dioxide.

Titanium dioxide (TiO2) displays photocatalytic behavior under near-ultraviolet (UV) illumination. In another scientific field, it is well understood that the excitation of localized plasmon polaritons on the surface of silver (Ag) nanoparticles (NPs) causes a tremendous increase of the near-field amplitude at well-defined wavelengths in the near UV. The exact resonance wavelength depends on the shape and the dielectric environment of the NPs. We expected that the photocatalytic behavior of TiO2 would be greatly boosted if it gets assisted by the enhanced near-field amplitudes of localized surface plasmon (LSP). Here we show that this is true indeed. We named this new phenomenon "plasmonic photocatalysis". The key to enable plasmonic photocatalysis is to deposit TiO2 on a NP comprising an Ag core covered with a silica (SiO2) shell to prevent oxidation of Ag by direct contact with TiO2. The most appropriate diameter for Ag NPs and thickness for the SiO2 shell giving rise to LSP in the near UV were estimated from Mie scattering theory. Upon implementing a device that took these design considerations into account, the measured photocatalytic activity under near UV illumination of such a plasmonic photocatalyst, monitored by decomposition of methylene blue, was enhanced by a factor of 7. The enhancement of the photocatalytic activity increases with a decreased thickness of the SiO2 shell. The plasmonic photocatalysis will be of use as a high performance photocatalyst in nearly all current applications but will be of particular importance for applications in locations of minimal light exposure.