Simple Binding and Dissociation of a Sialoglycoprotein Using Boronic Acid-Modified Functional Interfaces on Microparticles.

An overexpression of sialic acid is an indicator of metastatic cancer, and selective detection of sialic acid shows potential for cancer diagnosis. Boronic acid is a promising candidate for this purpose because of its ability to specifically bind to sialic acid under acidic conditions. Notably, the binding strength can be easily modulated by adjusting the pH, which allows for a simple dissociation of the bound sialic acid. In this study, we developed 5-boronopicolinic acid (5-BPA)-modified magnetic particles (BMPs) to selectively capture sialic acid biomolecules. We successfully captured fetuin, a well-known sialoglycoprotein, on BMPs at >104 molecules/particle using an acetate buffer (pH 5.0). Facile dissociation then occurred when the system was changed to a pH 7.6 phosphate buffer. This capture-and-release process could be repeated at least five times. Moreover, this system could enrich fetuin by more than 20 times. In summary, BMPs are functional particles for facile purification and concentration through the selective capture of sialic acid proteins and can improve detection sensitivity compared with conventional methods. This technology shows potential for the detection of sialic acid overexpression by biological particles.

Hydrogel capsule-based digital quantitative polymerase chain reaction.

Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however, is limited by the stability of droplets against thermal cycling. While the use of fluorinated oil or supplementation of surfactant could improve the stability of droplets, this process has also greatly increased the cost of ddPCR and limited post-PCR analysis. Here, we report a novel method known as gel capsule-based digital PCR (gc-dPCR) which includes a method to prepare hydrogel capsules encapsulating the PCR reaction mix, conducting PCR reaction, and readout by either quantitative PCR (qPCR) system or fluorescence microplate reader. We have compared the developed method to vortex ddPCR. Our approach results in higher fluorescence intensity compared to ddPCR suggesting higher sensitivity of the system. As hydrogel capsules are more stable than droplets in fluorinated oil throughout thermal cycling, all partitions can be quantified, thus preventing loss of information from low-concentration samples. The new approach should extend to all droplet-based PCR methods. It has greatly improved ddPCR by increasing droplets stability and sensitivity, and reducing the cost of ddPCR, which help to remove the barrier of ddPCR in settings with limited resources.

Improvement of Sensitivity and Speed of Virus Sensing Technologies Using nm- and µm-Scale Components.

Various viral diseases can be widespread and cause severe disruption to global society. Highly sensitive virus detection methods are needed to take effective measures to prevent the spread of viral infection. This required the development of rapid virus detection technology to detect viruses at low concentrations, even in the biological fluid of patients in the early stages of the disease or environmental samples. This review describes an overview of various virus detection technologies and then refers to typical technologies such as beads-based assay, digital assay, and pore-based sensing, which are the three modern approaches to improve the performance of viral sensing in terms of speed and sensitivity.

Gold Nanoparticles with Ligand/Zwitterion Hybrid Layer for Individual Counting of Influenza A H1N1 Subtype Using Resistive Pulse Sensing.

Resistive pulse sensing (RPS) is an analytical technique for detecting particles with nano- to micrometer diameters, such as proteins, viruses, and bacteria. RPS is a promising tool for diagnosis as it can analyze the characteristics of target particles individually from ion current blockades as pulse waveforms. However, it is difficult to discriminate analog targets because RPS merely provides physical information such as size, shape, concentration, and charge density of the analyte. Influenza A virus, which is 80-120 nm in diameter, has various subtypes, demonstrating the diversity of virus characteristics. For example, highly pathogenic avian influenza infections in humans are recognized as an emerging infectious disease with high mortality rates compared with human influenza viruses. Distinguishing human from avian influenza using their differing biological characteristics would be challenging using RPS. To develop a highly selective diagnostic system for infectious diseases, we combined RPS with molecular recognition. Gold nanoparticles (GNPs) that have human influenza A (H1N1 subtype) virus-specific sialic acid receptors on the surface were prepared as a virus label for RPS analysis. A sulfobetaine and sialic acid (ligand) hybrid surface was formed on the GNPs for the suppression of nonspecific interaction. The results show a size change of viruses derived from specific interactions with GNPs. In contrast, no size shift was observed when nonspecific sialic acid receptor-immobilized GNPs were used. Detection of viruses by individual particle counting could be a new facet of diagnosis.

Co-immobilized poly(ethylene glycol)-block-polyamines promote sensitivity and restrict biofouling on gold sensor surface for detecting factor IX in human plasma.

In order to detect an extremely low amount of human coagulation factor IX (FIX), poly(ethylene glycol) (PEG)/aptamer co-immobilized surface was constructed using original PEG-polyamine surface modification agents on surface plasmon resonance (SPR) sensor chip. Initially, a gold (Au) sensor chip of SPR was modified using poly(ethylene glycol)-b-poly[2-(N,N-dimethylamino)ethyl methacrylate] (PEG-b-PAMA) followed by treatment with SH-dT20 and was duplexed with anti-FIX aptamer extended using A24. Furthermore, the co-immobilization of pentaethylenehexamine-terminated poly(ethylene glycol) (N6-PEG) on the sensing surface completely quenched bio-fouling. On this dual tethered PEG-surface, we determined that the dissociation constant for FIX-aptamer interaction was 37 ± 10 pM, and the sensitivity of detection could reach up to 800 fM on using aptamer-FIX-antibody sandwich pattern detected by gold nanoparticle-conjugated anti-mouse antibody. We could detect FIX in the presence of abundant albumin. Furthermore, to mimic the actual detection of FIX in clinical samples, we demonstrated our experimental results with human blood plasma instead of FIX. Higher-sensitivity was attained because of dual polymers immobilized on Au surface, and this can emerge as a common strategy for any aptamer-protein interactions. The selective binding of aptamer in human blood plasma described here indicates the suitability of the present strategy for detection in clinically relevant samples.

High-performance surface acoustic wave immunosensing system on a PEG/aptamer hybridized surface.

Label-free immunoassay systems have the advantages of procedural simplicity and a low construction cost of surfaces for immunosensing. When label-free immunoassay systems are considered, the nonspecific adsorption of unwanted materials should be eliminated unless it aids in the detection of error. PEG is well-known as a blocking agent for the prevention of the adsorption of nonspecific binding materials when coimmobilized with ligands for targets such as antibodies and oligonucleotides. The construction strategy for PEG/ligand coimmobilized surfaces is an important point in the preparation of a high-performance assays because the physiological condition of the ligand depends strongly on its interaction with the PEG chain. In this report, we investigate the interaction between thrombin and a thrombin-binding aptamer (TBA) on a PEG/TBA coimmobilized surface by using a shear horizontal surface acoustic wave (SAW) sensor. The thrombin-TBA binding property shows remarkable differences with changes in the PEG density and the distance from the gold surface to the aptamer.

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.

Methodology to Detect Biological Particles Using a Biosensing Surface Integrated in Resistive Pulse Sensing.

Resistive pulse sensing (RPS) is an analytical method that can be used to individually count particles from a small sample. RPS simply monitors the physical characteristics of particles, such as size, shape, and charge density, and the integration of RPS with biosensing is an attractive theme to detect biological particles such as virus and bacteria. In this report, a methodology of biosensing on RPS was investigated. Polydopamine (PD), an adhesive component of mussels, was used as the base material to create a sensing surface. PD adheres to most materials, such as noble metals, metal oxides, semiconductors, and polymers; as a result, PD is a versatile intermediate layer for the fabrication of a biosensing surface. As an example of a biological particle, human influenza A virus (H1N1 subtype) was used to monitor translocation of particles through the pore membrane. When virus-specific ligands (6'-sialyllactose) were immobilized on the pore surface, the translocation time of the virus particles was considerably extended. The detailed translocation data suggest that the viral particles were trapped on the sensing surface by specific interactions. In addition, virus translocation processes on different pore surfaces were distinguished using machine learning. The result shows that the simple and versatile PD-based biosensor surface design was effective. This advanced RPS measurement system could be a promising analytical technique.

Gd@C82 metallofullerenes for neutron capture therapy-fullerene solubilization by poly(ethylene glycol)-block-poly(2-(N, N-diethylamino)ethyl methacrylate) and resultant efficacy in vitro.

Poly(ethylene glycol)-block-poly(2-(N,N-diethylamino)ethyl methacrylate) (PEG-b-PAMA) was found to solubilize fullerenes such as C60, and this technique was applied to metallofullerenes. Gd@C82 was easily dissolved in water in the presence of PEG-b-PAMA without any covalent derivatization, forming a transparent complex about 20-30 nm in diameter. Low cytotoxicity was confirmed in vitro. Neutron irradiation of cultured cells (colon-26 adenocarcinoma) with Gd@C82-PEG-b-PAMA-complexed nanoparticles showed effective cytotoxicity, indicating the effective emission of gamma rays and internal conversion electrons produced from the neutron capture reaction of Gd. This result suggests a potentially valuable approach to gadolinium-based neutron capture therapy.

Direct and label-free influenza virus detection based on multisite binding to sialic acid receptors.

A system to discriminate human or avian influenza A remains a highly sought-after tool for prevention of influenza pandemics in humans. Selective binding of the influenza A viral hemagglutinin (HA) to specific sialic acid (SA) receptors (Neu5Acα(2-6)Gal in humans, Neu5Acα(2-3)Gal in birds) is determined by the genotype of the HA and neuraminidase (NA) segments, making it one of the key characteristics that distinguishes human or avian influenza A virus. Here we demonstrate the direct detection of whole H1N1 influenza A virus using 6'-sialyllactose (Neu5Acα(2-6)Galß(1-4)Glc, 6SL)-immobilized gold electrodes as biosensing surfaces. The sensitivity was higher than that of conventional immunochromatographic technique (ICT) for influenza virus and not restricted by genetic drift. The label-free detection technology via direct attachment of a whole virus using a chemically modified electrode is a promising means to provide a simple and rapid diagnostic system for viral infections.

Application of surface enhanced Raman spectroscopy as a diagnostic system for hypersialylated metastatic cancers.

Early diagnosis of metastatic cancers could greatly limit the number of cancer-associated deaths. Aberrant surface expression of sialic acid (hypersialylation) on tumors correlating with metastatic incidence and its involvement in tumorigenesis and progression is widely reported; hence detection of hypersialylated tumors may be an effective strategy to identify metastatic cancers. We herein report on the application of phenylboronic acid-installed PEGylated gold nanoparticles coupled with Toluidine blue O (T/BA-GNPs) as SERS probes to target surface sialic acid (N-acetylneuraminic acid, Neu5Ac). Strong SERS signals from metastatic cancer cell lines (breast cancer; MDA-MB231 and colon cancer; Colon-26) were observed, contrary to non-metastatic MCF-7 cells (breast cancer). The detected SERS signals from various cancer cell lines correlated with their reported metastatic potential, implying that our T/BA-GNP based SERS system was capable of distinguishing the metastaticity of cells based on the surface Neu5Ac density. T/BA-GNP based SERS system could also significantly differentiate between hypersialylated tumor tissues and healthy tissues with high SERS signal to noise ratio, due to plasmon coupling between the specifically aggregated functionalized GNPs. Furthermore, we also confirmed reduction in SERS signals from MDA-MB231 surface upon treatment with our original reactive oxygen species (ROS)-scavenging polymeric micelle, nitroxide-radical containing nanoparticles (RNPs). The ROS-mediated abrogation of sialylation by impairing the activation of NF-κB-sialyltransferase signaling cascade upon RNP treatment was confirmed by expression studies and the T/BA-GNPs based SERS system. The aforementioned findings thus, establish T/BA-GNPs based SERS as a potential cytodiagnostic system to detect hypersialylated metastatic tumors and RNPs as anti-metastatic cancer drug candidates.

Specific Recognition of Human Influenza Virus with PEDOT Bearing Sialic Acid-Terminated Trisaccharides.

Conducting polymers are good candidates for biosensor applications when molecular recognition element is imparted. We developed trisaccharide-grafted conducting polymers for label-free detection of the human influenza A virus (H1N1) with high sensitivity and specificity. A 3,4-ethylenedioxythiophene (EDOT) derivative bearing an oxylamine moiety was electrochemically copolymerized with EDOT. The obtained film was characterized by cyclic voltammetry, X-ray photoelectron spectroscopy, scanning electron microscopy, stylus surface profilometer, and AC-impedance spectroscopy. The trisaccharides comprising Sia-α2,6'-Gal-Glu (2,6-sialyllactose) or Sia-α2,3'-Gal-Glu (2,3-sialyllactose) were covalently introduced to the side chain of the conducting polymers as a ligand for viral recognition. Immobilization of sialyllactose was confirmed by quartz crystal microbalance (QCM) and water contact angle measurements. Specific interaction of 2,6-sialyllactose with hemagglutinin in the envelope of the human influenza A virus (H1N1) was detected by QCM and potentiometry with enhanced sensitivity by 2 orders of magnitude when compared with that of commercially available kits. The developed conducting polymers possessing specific virus recognition are a good candidate material for wearable monitoring and point-of-care testing because of their processability and mass productivity in combination with printing technologies.

Use of boron cluster-containing redox nanoparticles with ROS scavenging ability in boron neutron capture therapy to achieve high therapeutic efficiency and low adverse effects.

A boron delivery system with high therapeutic efficiency and low adverse effects is crucial for a successful boron neutron capture therapy (BNCT). In this study, we developed boron cluster-containing redox nanoparticles (BNPs) via polyion complex (PIC) formation, using a newly synthesized poly(ethylene glycol)-polyanion (PEG-polyanion, possessing a (10)B-enriched boron cluster as a side chain of one of its segments) and PEG-polycation (possessing a reactive oxygen species (ROS) scavenger as a side chain of one of its segments). The BNPs exhibited high colloidal stability, selective uptake in tumor cells, specific accumulation, and long retention in tumor tissue and ROS scavenging ability. After thermal neutron irradiation, significant suppression of tumor growth was observed in the BNP-treated group, with only 5-ppm (10)B in tumor tissues, whereas at least 20-ppm (10)B is generally required for low molecular weight (LMW) (10)B agents. In addition, increased leukocyte levels were observed in the LMW (10)B agent-treated group after thermal neutron irradiation, and not in BNP-treated group, which might be attributed to its ROS scavenging ability. No visual metastasis of tumor cells to other organs was observed 1 month after irradiation in the BNP-treated group. These results suggest that BNPs are promising for enhancing the BNCT performance.

Sulfobetaine-terminated PEG improves the qualities of an immunosensing surface.

Poly(ethylene glycol) (PEG) possessing a sulfobetaine (SB) moiety at one end and a pentaethylenehexamine (N6) at the other end (SB-PEG-N6) was newly synthesized as a blocking agent for immunosensing surfaces. The N6 moiety strongly coordinates on gold surfaces, facilitating the tethering of the PEG chain to the sensor chip surface, and leaves the SB moiety free. Non-specific adsorption of bovine serum albumin (BSA) was analyzed on the SB-PEG-N6 tethered surface and compared with the methoxy-PEG-N6 (M-PEG-N6) tethered surface using a surface plasmon resonance (SPR) sensor. Non-specific BSA adsorption decreased with decreasing PEG chain length on the SB-PEG tethered chain surface. Non-specific adsorption of BSA decreased as ionic strength increased on SB-PEG-N6 surfaces; this phenomenon was completely opposite to that observed with an M-PEG-N6 tethered chain surface. The results show that SB moieties located close to the gold surface perform well with regard to protein rejection. Actually, low-molecular weight alkane thiol SB (SB-SH) showed minimum BSA adsorption. To evaluate protein recognition efficacy on a PEGylated surface, an antibody (IgG) immobilized surface was then constructed on a gold sensor chip using SB-PEG-N6 as the blocking agent. The specific protein recognition efficacy of SB-PEG-N6/IgG co-immobilized surfaces was higher than that obtained using SB-SH/IgG co-immobilized surfaces. We conclude that SB-terminated PEG exhibits the optimal qualities of a blocking agent, as it possesses both high suppression efficacy of nonspecific protein adsorption and specific protein recognition ability.

Intra-tumor distribution of metallofullerene using micro-particle induced X-ray emission (PIXE).

To clarify the intra tumor distribution of gadlinium containing fullerene (Gd@C82), micro particle induced X-ray emission (Micro-PIXE) analysis were performed. The tumor bearing BALB/c mice were injected Gd@C82 and subcutaneous tumors were taken from 48h after the intravenous injection. Using the Micro-PIXE method, we could visualize Gd intra tumor distribution. Therefore our results indicate the possibility that Micro-PIXE is useful technique for imaging the bioditribution of Gd, and Gd@C82 is potentially useful Gd carrier for NCT.

Tumor growth suppression by gadolinium-neutron capture therapy using gadolinium-entrapped liposome as gadolinium delivery agent.

Neutron capture therapy (NCT) is a promising non-invasive cancer therapy approach and some recent NCT research has focused on using compounds containing gadolinium as an alternative to currently used boron-10 considering several advantages that gadolinium offers compared to those of boron. In this study, we evaluated gadolinium-entrapped liposome compound as neutron capture therapy agent by in vivo experiment on colon-26 tumor-bearing mice. Gadolinium compound were injected intravenously via tail vein and allowed to accumulate into tumor site. Tumor samples were taken for quantitative analysis by ICP-MS at 2, 12, and 24 h after gadolinium compound injection. Highest gadolinium concentration was observed at about 2 h after gadolinium compound injection with an average of 40.3 µg/g of wet tumor tissue. We performed neutron irradiation at JRR-4 reactor facility of Japan Atomic Energy Research Institute in Tokaimura with average neutron fluence of 2×10¹² n/cm². The experimental results showed that the tumor growth suppression of gadolinium-injected irradiated group was revealed until about four times higher compared to the control group, and no significant weight loss were observed after treatment suggesting low systemic toxicity of this compound. The gadolinium-entrapped liposome will become one of the candidates for Gd delivery system on NCT.

Pharmacokinetics of core-polymerized, boron-conjugated micelles designed for boron neutron capture therapy for cancer.

Core-polymerized and boron-conjugated micelles (PM micelles) were prepared by free radical copolymerization of a PEG-b-PLA block copolymer bearing an acetal group and a methacryloyl group (acetal-PEG-b-PLA-MA), with 1-(4-vinylbenzyl)-closo-carborane (VB-carborane), and the utility of these micelles as a tumor-targeted boron delivery system was investigated for boron neutron capture therapy (BNCT). Non-polymerized micelles (NPM micelles) that incorporated VB-carborane physically showed significant leakage of VB-carborane (ca. 50%) after 12 h incubation with 10% fetal bovine serum (FBS) at 37 °C. On the other hand, no leakage from the PM micelles was observed even after 48 h of incubation. To clarify the pharmacokinetics of the micelles, (125)I (radioisotope)-labeled PM and NPM micelles were administered to colon-26 tumor-bearing BALB/c mice. The (125)I-labeled PM micelles showed prolonged blood circulation (area under the concentration curve (AUC): 943.4) than the (125)I-labeled NPM micelles (AUC: 495.1), whereas tumor accumulation was similar for both types of micelles (AUC(PM micelle): 249.6, AUC(NPM micelle): 201.1). In contrast, the tumor accumulation of boron species in the PM micelles (AUC: 268.6) was 7-fold higher than the NPM micelles (AUC: 37.1), determined by ICP-AES. Thermal neutron irradiation yielded tumor growth suppression in the tumor-bearing mice treated with the PM micelles without reduction in body weight. On the basis of these data, the PM micelles represent a promising approach to the creation of boron carrier for BNCT.

Photothermal reshaping of gold nanorods depends on the passivating layers of the nanorod surfaces.

Photothermal reshaping of gold nanorods was triggered by pulsed-laser irradiation. The efficiency of the reshaping was strongly dependent on the surface conditions of the gold nanorods. When the gold nanorods were dispersed in concentrated hexadecyltrimethylammonium bromide (CTAB), the gold nanorods were efficiently transformed into a phi-shape. By comparison when poly(styrene sulfonate), poly(vinylpyrrolidone), poly(ethylene glycol), or phosphatidylcholine layers were used, the CTAB layers were found to be a better thermal insulator that helped to enhance the photothermal reshaping of the gold nanorods.