Yunxian Piao

Three-dimensional graphene micropillar based electrochemical sensor for phenol detection

A three-dimensional (3D) graphene incorporated electrochemical sensor was constructed for sensitive enzyme based phenol detection. To form the 3D graphene structure, polydimethylsiloxane (PDMS) micropillars were fabricated in the microchannel by using a conventional photolithography and the surface was modified with 3-aminopropyltriethoxysilane. Then, the negatively charged graphene oxide sheets were electrostatically adsorbed on the PDMS micropillar surface, and reduced in the hydrazine vapor. The resultant 3D graphene film provides a conductive working electrode as well as an enzyme-mediated sensor with a large surface area. After bonded with an electrode patterned glass wafer, the 3D graphene based electrochemical sensor was produced. Using the 3D graphene as a working electrode, an excellent electron transfer property was demonstrated by cyclic voltammetry measurement in an electrolyte solution containing 1mM K3Fe(CN)6 and 0.1 M KCl. To utilize the 3D graphene as an enzyme sensor, tyrosinase enzymes were immobilized on the surface of the graphene micropillar, and the target phenol was injected in the microchannel. The enzyme catalytic reaction process was monitored by amperometric responses and the limit of detection for phenol was obtained as 50 nM, thereby suggesting that the 3D graphene micropillar structure enhances the enzyme biosensing capability not only by increasing the surface area for enzyme immobilization, but also by the superlative graphene conductivity property.

Multiplexed immunoassay using the stabilized enzymes in mesoporous silica

Multiplexed immunoassay system was developed using the enzyme-immobilized mesoporous silica in a form of nanoscale enzyme reactors (NERs), which improve the enzyme loading, activity, and stability. Glucose oxidase (GO) and trypsin (TR) were adsorbed into mesoporous silica and further crosslinked for the construction of NERs, and antibody-conjugated NERs were employed for the analysis of target antigens in a sandwich-type magnetic bead-based immunoassay. This approach, called as NER-LISA (NER-linked immunosorbent assay), generated signals out of enzyme reactions that correlated well with the concentration of target antigens. The detection limit of NER-LISA using NER-GO and anti-human IgG was 67pM human IgG, and the sensitivity was 20 times higher than that of the conventional ELISA using anti-human IgG conjugated GO. Antibody-conjugated NER-GO and NER-TR were successfully employed for the simultaneous detection of two target antigens (human IgG and chicken IgG) in a solution by taking advantage of signals at different wavelengths (absorbances at 570nm and 410nm, respectively) from the assays of GO and TR activities, demonstrating the potential of NER-LISA in multiplexed immunoassay. The NER-LISA approach also enabled the successful use of a protease (trypsin), because the NER approach can effectively retain the protease molecules within the mesoporous silica and prevent the digestion of antibodies and enzymes during the whole process of NER-LISA.

Sensitive and high-fidelity electrochemical immunoassay using carbon nanotubes coated with enzymes and magnetic nanoparticles

We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months.

Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel

The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.

A Novel Molecular Beacon Bearing a Graphite Nanoparticle as a Nanoquencher for In situ mRNA Detection in Cancer Cells

Molecular beacons (MBs) have shown fascinating applications in many biological fields. However, exploration of cost-effective, sensitive, stable and efficient MB for in situ live cell- based assay has still room for improvement. In this regards, we have developed a novel MB which bears a spherical graphite nanoparticle (GN) as a fluorescent quencher. The GN resulted in the high quenching efficiency, and the presence of GN enhanced the biological stability and transfection of the MB into the cells, thereby enabling the real-time survivin mRNA detection and quantification in the MCF-7 breast cancer cells. These results demonstrated that the advancement of the proposed MB containing a GN nanoquencher can be used as a robust molecular probe for genetic analysis in the cells.

A highly sensitive immunoassay using antibody-conjugated spherical mesoporous silica with immobilized enzymes.

A highly sensitive immunoassay was developed by using antibody-conjugated spherical mesoporous silica with immobilized enzymes. The higher ratio of enzyme/antibody than conventional ELISA improved both the sensitivity and dynamic range. Especially, the use of spherical mesoporous silica could achieve a limit of detection (LOD) with a sensitivity that is 20 times more than that of ELISA using amorphous silica.

Fabrication of various cross-sectional shaped polymer microchannels by a simple PDMS mold based stamping method

We describe a facile and simple stamping method to fabricate various cross-sectional shaped microfluidic channels in transparent polymer materials such as polydimethylsiloxane (PDMS), poly(methylmethacrylate) (PMMA), polystyrene (PS), and cyclic olefin copolymer (COC). First, microchannels of circular, rectangular, and triangular shape and different size were fabricated on a silicon wafer through an isotropic or anisotropic wet etching process, and the resultant microchannels on silicon wafers were transferred to PDMS through a replication technique. The produced PDMS with replicated convex microchannels served as a master mold. A variety of polymer solutions were pressed down against the PDMS master mold, and cured until the solvent was evaporated, generating circular, rectangular, and triangular shaped PDMS, PMMA, PS, and COC microchannels. The microchannels could be repeatedly prepared with a narrow standard deviation, thus demonstrating high reproducibility of the proposed method. The microchannel dimensions, shape, and surface roughness could be controlled by tuning the channel width of the mask, the wet etching direction, the etchant solution, and the concentration of KOH, respectively, when the microchannels were fabricated on a silicon wafer. This simple but efficient PDMS mold based stamping method can be widely used for fabricating different shaped microchannels on diverse polymer materials with high reproducibility, low cost, and high speed.

Generation of hierarchical nano- and microwrinkle structure for smooth muscle cell alignment

On the contrary to the complicated and high-cost photolithography based topographical patterning, the non-lithographical strain responsive wrinkling method can generate a variety of wrinkle structures with ease, high uniformity, and cost-effectiveness. The strain responsive wrinkling approach relied on the modulus difference between a thin and stiff film and a soft substrate, resulting in a periodic out-of-plane buckling deformation upon the release of the compressive stress. While the previous reports were dedicated to the micropattern generation, we, in this study, investigated the effect of the UV/O exposure time (10, 20, 30, 40, 50, and 60 min) and the stretching rate (10, 20, 30, and 40%) of the PDMS substrate on the wavelength and the amplitude of the wrinkle patterns in details. Sole nanowrinkle as well as hierarchical nano/microwrinkle patterns could be fabricated through the fine control of those factors. Furthermore, we examined the human aortic smooth muscle cell (HASMCs) alignment on the topographical patterned surface, and found that more than 80% of the HASMCs were cultured and aligned well along with the hierarchical nano/microwrinkle rather than the nanowrinkle pattern.