Investigation of the Nanoparticulation Method and Cell-Killing Effect following the Mitochondrial Delivery of Hydrophobic Porphyrin-Based Photosensitizers.

Photodynamic therapy is expected to be a less invasive treatment, and strategies for targeting mitochondria, the main sources of singlet oxygen, are attracting attention to increase the efficacy of photodynamic therapy and reduce its side effects. To date, we have succeeded in encapsulating the photosensitizer rTPA into MITO-Porter (MP), a mitochondria-targeted Drug Delivery System (DDS), aimed at mitochondrial delivery of the photosensitizer while maintaining its activity. In this study, we report the results of our studies to alleviate rTPA aggregation in an effort to improve drug efficacy and assess the usefulness of modifying the rTPA side chain to improve the mitochondrial retention of MITO-Porter, which exhibits high therapeutic efficacy. Conventional rTPA with anionic side chains and two rTPA analogs with side chains that were converted to neutral or cationic side chains were encapsulated into MITO-Porter. Low-MP (MITO-Porter with Low Drug/Lipid) exhibited high drug efficacy for all three types of rTPA, and in Low-MP, charged rTPA-encapsulated MP exhibited high drug efficacy. The cellular uptake and mitochondrial translocation capacities were similar for all particles, suggesting that differences in aggregation rates during the incorporation of rTPA into MITO-Porter resulted in differences in drug efficacy.

Antigen/Adjuvant-Displaying Enveloped Viral Replica as a Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidate.

We report a promising cancer vaccine candidate comprising antigen/adjuvant-displaying enveloped viral replica as a novel vaccine platform. The artificial viral capsid, which consists of a self-assembled ß-annulus peptide conjugated with an HER2-derived antigenic CH401 peptide, was enveloped within a lipid bilayer containing the lipidic adjuvant α-GalCer. The use of an artificial viral capsid as a scaffold enabled precise control of its size to ∼100 nm, which is generally considered to be optimal for delivery to lymph nodes. The encapsulation of the anionically charged capsid by a cationic lipid bilayer dramatically improved its stability and converted its surface charge to cationic, enhancing its uptake by dendritic cells. The developed CH401/α-GalCer-displaying enveloped viral replica exhibited remarkable antibody-production activity. This study represents a pioneering example of precise vaccine design through bottom-up construction and opens new avenues for the development of effective vaccines.

Dimethoxytriadinylation LC-MS/MS of Novichok A-Series Degradation Products in Human Urine.

Novichok A-series compounds, novel nerve agents, pose an increasing threat to citizens worldwide; however, no analytical methods have been reported for detecting their hydrolysis products. Herein, a screening method was developed to detect and identify Novichok A-series degradation products (hydrolysates of A230, A232, A234, A262, and one related compound) and alkyl methylphosphonic acids (RMPAs, conventional nerve agent hydrolysates) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified a suitable derivatization reagent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), and optimized the reaction conditions. The derivatized esters of Novichok A-series degradation products were stable and easily detected. We used this derivatization to achieve the first analytical method for Novichok hydrolysis products in urine (0.40-4.0 ng/mL). The detection limits of the RMPAs (0.1-0.4 ng/mL) were comparable to those presented in previous reports involving pentafluorobenzylation or direct LC-MS/MS. The applicability of the newly developed method was evaluated by analyzing urine samples from the OPCW Fifth Biomedical Proficiency Test.

Topology-Dependent Interaction of Cyclic Poly(ethylene glycol) Complexed with Gold Nanoparticles against Bovine Serum Albumin for a Colorimetric Change.

Unique physical and chemical properties arising from a polymer topology recently draw significant attention. In this study, cyclic poly(ethylene glycol) (c-PEG) was found to significantly interact with bovine serum albumin (BSA), suggested by nuclear magnetic resonance, dynamic light scattering, and fluorescence spectroscopy. On the other hand, linear HO-PEG-OH and MeO-PEG-OMe showed no affinity. Furthermore, a complex of gold nanoparticles and c-PEG (AuNPs/c-PEG) attracted BSA to form aggregates, and the red color of the AuNPs dispersion evidently disappeared, whereas ones with linear PEG or without PEG did not demonstrate such a phenomenon. The interactions among BSA, AuNPs, and PEG were investigated by changing the incubation time and concentration of the components by using UV-Vis and fluorescence spectroscopy.

Enzyme kinetics in confined geometries at the single enzyme level.

The effects of increased confinement on the catalytic rates of individual enzyme molecules were studied at the single molecule level using femtolitre chambers and molecular crowders. According to the increase of confinement, from micro to nanometer cubic space in the chambers, the hydrolysis rate of ß-galactosidase (ß-gal) decreased to one-tenth of the rate in bulk. When molecular crowders suppressed the diffusion rates that reduced the collision chance of an enzyme and a substrate, the hydrolysis rate also decreased, which happened also in the case of femtolitre chambers. However, their kinetic trend was different especially from the viewpoint of the diffusion rates in diffusion-limited space. These data suggested that cell or organelle-scale confined environments might affect the kinetics of biochemical reactions and emphasized the importance of understanding enzyme kinetics in the in vivo environment.

Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 µL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.

Observation of Ethanol-Induced Condensation and Decondensation Processes at a Single-DNA Molecular Level in Microfluidic Devices Equipped with a Rapid Solution Exchange System.

Conformational transitions from secondary (e.g., B- to A-form DNA) to higher-order (e.g., coil to globule) transitions play important roles in genome expression and maintenance. Several single-molecule approaches using microfluidic devices have been used to determine the kinetics of DNA chromatin assembly because microfluidic devices can afford stretched DNA molecules through laminar flow and rapid solution exchange. However, some issues, particularly the uncertainty of time 0 in the solution exchange process, are encountered. In such kinetic experiments, it is critical to determine when the target solution front approaches the target DNA molecules. Therefore, a new design for a microfluidic device is developed that enables the instantaneous exchange of solutions in the observation channel, allowing accurate measurements of DNA conformational transitions; stepwise, ethanol-induced conformational transitions are revealed. Although full DNA contraction from coil to globule is observed with >50% ethanol, no outstanding change is observed at concentrations <40% in 10 min. With 50% ethanol solution, the DNA conformational transition passes through two steps: (i) fast and constant-velocity contraction and (ii) relatively slow contraction from the free end. The first process is attributed to the B to A conformational transition by gradual dehydration. The second process is due to the coil-globule transition as the free end of DNA starts the contraction. This globular structure formation counteracts the shear force from the microfluids and decelerates the contraction velocity. This real-time observation system can be applied to the kinetic analysis of DNA conformational transitions such as kinetics of chromatin assembly and gene expression.

Noncompetitive Fluorescence Polarization Immunoassay for Protein Determination.

Fluorescent polarization immunoassay (FPIA) is a single-step immunoassay method that is applicable to point-of-care testing; however, its applicability to large biomolecules has been restricted because ordinary FPIA is a competitive assay. Here, we report a noncompetitive FPIA using the variable domain from the heavy chain of a camelid antibody (VHH antibody). FPIA with VHH was successfully used to quantitate rabbit immunoglobulin G (IgG) and demonstrated a wider response range than that observed with antibody-binding (Fab) fragment. Then, using a portable FPIA instrument, a VHH-based immunoassay of human IgG in a human serum certified reference material was demonstrated.

The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing on Their Lymph Node Transitivity and Distribution.

Because the lymph node (LN) is a critical organ for inducing immune responses against pathogens and cancers, the transport of immune functional molecules such as antigens and adjuvants to LNs by delivery systems is a useful strategy for the effective outcome of an immune response. The size and charge of a delivery system largely affect the transitivity to and distribution within LN. Although pH-sensitive lipid nanoparticles (LNPs) prepared by microfluidic mixing are the latest delivery system to be applied clinically, the effects of their size and charge on the transitivity to and distribution within LN are currently unknown. We investigated the size and charge effect of LNPs prepared by microfluidic mixing on transitivity to and distribution within LNs. A 30 nm-sized LNP (30-LNP) was efficiently translocated to LNs and was taken up by CD8+ dendritic cells, while the efficiency was drastically decreased in the cases of 100 and 200 nm-sized LNPs. Furthermore, a comparative study between neutral, positively, and negatively charged 30-LNP revealed that the negative 30-LNP moved to the LN more efficiently than the other LNPs. Interestingly, the negative 30-LNP reached the deep cortex, namely, the T cell zone. Our findings provide informative insights for designing LN-targeting LNPs prepared by microfluidic mixing and for the translocation of nanoparticles in LNs.

Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer.

A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIV hemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1-H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 µL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.

Ultrasensitive detection of disease biomarkers using an immuno-wall device with enzymatic amplification.

We present an ultrasensitive immunoassay system for disease biomarkers utilizing the immuno-wall device and an enzymatic amplification reaction. The immuno-wall device consisted of 40 microchannels, each of which contained an antibody-modified wall-like structure along the longitudinal axis of the microchannel. The wall was fabricated with a water-soluble photopolymer containing streptavidin by photolithography, and biotinylated capture antibodies were immobilized on the sides through streptavidin-biotin interaction. For an assay, introducing the target biomarker and secondary and labeled antibodies produced a sandwich complex anchored on the sides of the wall. A conventional immuno-wall device uses a fluorescence-labeled antibody as a labeling antibody. To achieve an ultrasensitive detection of a trace biomarker, we used an enzyme label and amplified the signal with the enzymatic reaction with a fluorogenic substrate in the microchannel. The highest signal/background ratio was obtained by using alkaline phosphatase-labeled antibody and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate. To evaluate the device performance, we detected human C-reactive protein (CRP) as a model biomarker. The detection limit (LOD) of CRP in phosphate-buffered saline was 2.5 pg mL-1 with a sample volume of 0.25 µL. This LOD was approximately 3 orders of magnitude lower than that obtained with fluorescent-dye (DyLight 650)-labeled antibody. In addition, the present device provided a wide detection range of 0.0025-10 ng mL-1 for CRP. We successfully developed an ultrasensitive immunoassay system with simple operation and only a small sample volume.

A competitive immunoassay system for microfluidic paper-based analytical detection of small size molecules.

The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (µPADs) is reported. The µPADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) was deposited at the control and test zones. µPAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody to travel past the capture zone and into the test zone via capillary action. The developed competitive immunoassay system was successfully demonstrated on the µPAD detection of biotin as a model compound with a detection limit of 0.10 µg mL-1. The applicability of the proposed immunoassay system for point-of-need testing was further demonstrated using aflatoxin B1, a highly toxic foodborne substance, with a detection limit of 1.31 ng mL-1. The µPAD with the competitive immunoassay format showed promising results for practical applications in point-of-need testing involving small molecular weight targets in food and water safety and quality monitoring, environmental analysis, and clinical diagnostics.

Image analysis for a microfluidic paper-based analytical device using the CIE L*a*b* color system.

The combination of a microfluidic paper-based analytical device (µPAD) and digital image analysis is widely used for quantitative analysis with µPADs because of its easy and simple operation. Herein, we have demonstrated a quantitative analysis based on multiple color changes on a µPAD. The CIE L*a*b* color system was employed to analyse the digital images obtained with the µPAD. We made pH measurements using a universal pH-indicator showing multiple color changes for various pH values of aqueous test solutions. The detectable pH range of this method was wider than the typical grayscale-based image analysis, and we succeeded in the measurements for a wide pH range of 2-9.

Novel concept of washing for microfluidic paper-based analytical devices based on capillary force of paper substrates.

A novel washing technique for microfluidic paper-based analytical devices (µPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow µPADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model µPAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 µg mL-1. Graphical Abstract A novel washing technique for microfluidic paper-based analytical devices (µPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported.

An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas.

World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69-80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene (IDH1), of which 83-90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 µl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice.

Arrangement of a nanostructure array to control equilibrium and nonequilibrium transports of macromolecules.

Exploiting the nonequilibrium transport of macromolecules makes it possible to increase the separation speed without any loss of separation resolution. Here we report the arrangement of a nanostructure array in microchannels to control equilibrium and nonequilibrium transports of macromolecules. The direct observation and separation of macromolecules in the nanopillar array reported here are the first to reveal the nonequilibrium transport, which has a potential to overcome the intrinsic trade-off between the separation speed and resolution.

Fluorescence polarization measurement system using a liquid crystal layer and an image sensor.

The detection system which enables simultaneous fluorescence polarization (FP) measurement of multiple samples was proposed and proven by a proof-of-concept experiment on the viscosity dependence of FP of fluorescein sample in water-ethylene glycol solution and another experiment on the FP immunoassay of prostaglandin E2 sample. The measurement principle of FP is based on the synchronization between the orientation of the liquid crystal molecules and the sampling frequency of a CCD. This report is the first description of the simultaneous FP measurement of multiple samples. This system has a great potential for equipment miniaturization and price reduction as well as providing simultaneous FP measurement of multiple samples.

A method of cryoprotection for protein crystallography by using a microfluidic chip and its application for in situ X-ray diffraction measurements.

We demonstrate a seamless and contactless method from protein crystallization to X-ray analysis using a microfluidic chip with the aim of obtaining a complete crystallographic data set of a protein crystal under cryogenic conditions. Our microfluidics-based approach did not require direct manipulation of the protein crystal. Therefore, the microfluidic chip approach is suitable for novices of X-ray analysis of protein crystals. We also investigated the effect of stepwise cryoprotection on the quality of protein crystals. Protein crystals with cryoprotection via on-chip manipulation did not show deterioration of crystallographic quality of the protein crystal. The complete diffraction data set of a protein crystal, which is required for determining the 3D structure of the target protein, is obtainable by a simple manipulation.

An instrument-free, screen-printed paper microfluidic device that enables bio and chemical sensing.

This paper describes a simple and instrument-free screen-printing method to fabricate hydrophilic channels by patterning polydimethylsiloxane (PDMS) onto chromatography paper. Clearly recognizable border lines were formed between hydrophilic and hydrophobic areas. The minimum width of the printed channel to deliver an aqueous sample was 600 µm, as obtained by this method. Fabricated microfluidic paper-based analytical devices (µPADs) were tested for several colorimetric assays of pH, glucose, and protein in both buffer and artificial urine samples and results were obtained in less than 30 min. The limits of detection (LODs) for glucose and bovine serum albumin (BSA) were 5 mM and 8 µM, respectively. Furthermore, the pH values of different solutions were visually recognised with the naked eye by using a sensitive ink. Ultimately, it is expected that this PDMS-screen-printing (PSP) methodology for µPADs can be readily translated to other colorimetric detection and hydrophilic channels surrounded by a hydrophobic polymer can be formed to transport fluids toward target zones.