Developing centrifugal force real-time digital PCR for detecting extremely low DNA concentration.

Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/µL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis.

Multifunctional hand-held sensor using electronic components embedded in smartphones for quick PCR screening.

We focused on the development of a hand-held pathogen-detection device using smartphone-embedded electronic elements combined with functionalized magnetic particles (MPs) and sepharose. To perform affinity chromatography for evaluating DNA amplicons, avidin-conjugated MPs and succinimide-linked sepharose were used with biotin-primers. To mimic the centrifugal-based affinity ligand chromatography, a smartphone-mountable low-power fan was plugged into the charging port of a smartphone. The charging port stably emitted electric current at 3.0 V, and the fan blades were modified for use as a portable rotor. Based on the binding variation of MPs with DNA amplicons, the position of MPs in sepharose changed significantly during centrifugation. The change in distance was optically analyzed using the illumination sensor of the smartphone with respect to the altered transmittance due to the MPs. Amplified genes from Escherichia. coli O157:H7 samples ranging from 1.0 × 101 to 1.0 × 106 colony-forming units could be rapidly and immediately detected by the naked eye using a simple smartphone-based optical device. The results indicated that this novel biosensing technique is suitable for use as a point-of-care testing device in both industrial and clinical fields.

Naked eye detection of an amplified gene using metal particle-based DNA transport within functionalized porous interfaces.

The current study focuses on developing a system for visually detecting an amplified bacterial (Escherichia coli O157:H7) gene using a heavy metal particle (MP) and functionalized porous sepharose gel. To functionalize DNA-specificity to the MP, an avidin-modified MP was employed in combination with a biotin-conjugated primer. The porous sepharose matrix was functionalized with an amine-reactive group, such as N-hydroxysuccinimide (NHS), to achieve separation upon binding of the amplified gene. The pristine avidin-MPs strongly react with NHS-sepharose via imide bonds owing to the exposure of the amine group on the avidin-MP surface. Conversely, together with the amplified gene, the avidin-MPs are relatively less interactive toward the sepharose gel by steric hindrance of the amplified gene toward the imide bond between NHS and the amine groups. Owing to the higher molecular mass of the MP, those metal particles complexed with the amplified gene pass through the sepharose matrix when centrifugal force is applied. The MPs that are thus separated can be easily visualized by the naked eye owing to their inherent reddish-brown color. A polymerase chain reaction (PCR) product of E. coli O157:H7, present in concentrations ranging from 1.0 × 101 to 1.0 × 106 colony forming units (CFU), in actual food sample was evaluated with high sensitivity and reproducibility. We expect that the MP-based sensing system, which allows for visual detection of PCR-amplified genes, can be clinically used as a point-of-care testing device.

Development of Lateral Flow Assay Based on Size-Controlled Gold Nanoparticles for Detection of Hepatitis B Surface Antigen.

In this study, we developed lateral flow assay (LFA) biosensors for the detection of hepatitis B surface antigens using well-controlled gold nanoparticles (AuNPs). To enhance colorimetric signals, a seeded growth method was used for the preparation of size-controlled AuNPs with a narrow size distribution. Different sizes of AuNPs in the range of 342-137.8 nm were conjugated with antibodies and then optimized for the efficient detection of LFA biosensors. The conjugation stability was investigated by UV-vis spectroscopy of AuNP dispersion at various pH values and concentrations of antibody. Based on optimized conjugation conditions, the use of 42.7 ± 0.8 nm AuNPs exhibited superior performance for the detection of LFAs relative to other sizes of AuNPs.

Bio-inspired Hierarchical Nanowebs for Green Catalysis.

Bio-inspired 3D hierarchical nanowebs are fabricated using silicon micropillars, carbon nanotubes (CNT), and manganese oxide. The Si pillars act as artificial branches for growing CNTs and the secondary metal coating strengthens the structures. The simple but effective structure provides both chemical and mechanical stability to be used as a green catalyst for recycling waste polymers into raw materials.

Ultrasonic bonding method for heterogeneous microstructures using self-balancing jig.

Perfect sealing of heterogeneous microstructures in plastic-based microfluidic devices is a significant and urgent challenge to be able to apply them in various microfluidic-based applications, including biosensing, biofiltering, chemical reactors and lab-on-a-chip. In this study we report a simple but practical and effective method to bond a microstructure-incorporated microfluidic device using an ultrasonic bonding method. The specially designed hemisphere-shaped jig, which is called a self-balancing jig, provides a free motion in all x, y, and z directions. These unique properties of the jig allow us to precisely adjust and bond the heterogeneous microstructures in the device. A conventional jig shows in solution leakages around the heterogeneous microstructures while the self-balancing jig did not show any leakages in devices. Furthermore, the bonding performance was also confirmed by using a black ink and fluorescent dye solution. The micro-pillar arrays in the device also demonstrated its capability for selective filtering of microbeads. We believe that this technique would be a useful tool for producing microfluidic devices with heterogeneous microstructures.

Sonochemical-assisted synthesis of 3D graphene/nanoparticle foams and their application in supercapacitor.

Graphene and its derivatives have attracted much attention in application of electrochemical devices. Construction of three-dimensional (3D) heterostructured composites is promising for establishing high-performance devices, which enables large surface area, facilitated ion and electron transport, and synergistic effects between multicomponents. Here, we report a simple and general sonochemical-assisted synthesis to prepare various 3D porous graphene/nanoparticle (i.e., Pt, Au, Pd, Ru, and MnO2) foams using colloidal template. The 3D porous network structure of composite foams significantly improves a large surface area of around 550m(2)g(-1) compared to the bare graphene (215m(2)g(-1)). This unique structure of 3D graphene/MnO2 enables further improvement of electrochemical characteristics, compared with bare graphene/MnO2 composite, showing a high specific capacitance of 421Fg(-1) at 0.1Ag(-1), high rate capability (97% retention at 20Ag(-1)), and good cycling performance (97% retention over 1000 cycles). Moreover, electrochemical impedance analysis demonstrates that electron and ion transfer are triggered by 3D porous structure.

Micropillar arrays enabling single microbial cell encapsulation in hydrogels.

Single microbial cell encapsulation in hydrogels is an important task to find valuable biological resources for human welfare. The conventional microfluidic designs are mainly targeted only for highly dispersed spherical bioparticles. Advanced structures should be taken into consideration for handling such aggregated and non-spherical microorganisms. Here, to address the challenge, we propose a new type of cylindrical-shaped micropillar array in a microfluidic device for enhancing the dispersion of cell clusters and the isolation of individual cells into individual micro-hydrogels for potential practical applications. The incorporated micropillars act as a sieve for the breaking of Escherichia coli (E. coli) clusters into single cells in a polymer mixture. Furthermore, the combination of hydrodynamic forces and a flow-focusing technique will improve the probability of encapsulation of a single cell into each hydrogel with a broad range of cell concentrations. This proposed strategy and device would be a useful platform for genetically modified microorganisms for practical applications.

A universal spring-probe system for reliable probing of electrochemical lab-on-a-chip devices.

For achieve sensitivity in lab-on-a-chip electrochemical detection, more reliable probing methods are required, especially for repeated measurements. Spring-probes are a promising candidate method which can replace needle-like probes and alligator clips that usually produce scratches on the surface of gold electrodes due to the strong physical contacts needed for electrochemical measurements. The superior reliability of amperometric measurements by a spring-probe system was compared with results by conventional probing methods. We demonstrated that a universal spring-probe system would be potentially suitable to achieve high performance in lab-on-a-chip devices using electrochemical detection.

Microbial inactivation and pesticide removal by remote exposure of atmospheric air plasma in confined environments.

Microbial inactivation and pesticide removal by remote exposure of atmospheric air plasma were investigated in confined environments, including an airtight box and commercial refrigerator. The relative sterilization ratios of remote plasma exposure in an airtight box were found to be affected by the distance from the plasma generator, the volume of box and the time of irradiation; however, over 99% saturation was obtained within only 120 s in all experiments. The sterilization of microorganisms and the removal of pesticide in a refrigerator with a volume of 292 l were also successfully achieved, resulting in over 99% inactivation or decontamination in a few minutes. Considering the reported results by direct plasma exposure and circulation, it can be concluded that the confined environment enhances the efficient irradiation of plasma by eliminating air flow. This system can be applied to the storage to keep agricultural products freshly and exclusion of harmful materials on the products.