Highly porous core-shell polymeric fiber network.

Core-shell nanofibers are of great interest in the field of tissue engineering and cell biology. We fabricated porous core-shell fiber networks using an electrospinning system with a water-immersed collector. We hypothesized that the phase separation and solvent evaporation process would enable the control of the pore formation on the core-shell fiber networks. To synthesize porous core-shell fiber networks, we used polycaprolactone (PCL) and gelatin. Quantitative analysis showed that the sizes of gelatin-PCL core-shell nanofibers increased with PCL concentrations. We also observed that the shapes of the pores created on the PCL fiber networks were elongated, whereas the gelatin-PCL core-shell fiber networks had circular pores. The surface areas of porous nanofibers were larger than those of the nonporous nanofibers due to the highly volatile solvent and phase separation process. The porous core-shell fiber network was also used as a matrix to culture various cell types, such as embryonic stem cells, breast cancer cells, and fibroblast cells. Therefore, this porous core-shell polymeric fiber network could be a potentially powerful tool for tissue engineering and biological applications.

Characteristics of a liposome immunoassay on a poly(methyl methacrylate) surface.

Liposome immunoassay (LIA) is based on enzyme immunoassay (EIA) but the detection sensitivity could be significantly enhanced by using antibody-coupled immunoliposomes encapsulating HRP (horse radish peroxidase). Here, we applied LIA to non-porous poly(methyl methacrylate) (PMMA) and polystyrene (PS) surfaces to compare its detection sensitivity with that of EIA, using rabbit IgG (a ligand molecule) and anti-rabbit IgG antibody (a capture molecule) as the model system. LIA developed much stronger color signals than EIA, especially at a lower concentration range (< ca. 1 microg mL(-1)). PMMA showed higher affinity toward rabbit IgG than the PS surface, and the anti-rabbit IgG antibody adsorbed on PMMA was more stable than that on PS. Furthermore, the effects of spot volume and antibody concentration on the signal density were analyzed. The signal density increased as the antibody concentration increased, but it was not significantly affected by the spot volume (2.5-20 microL). In conclusion, LIA on PMMA as a solid support is a very useful, highly sensitive microarray detection system.

Photolithographic fabrication of poly(ethylene glycol) microstructures for hydrogel-based microreactors and spatially addressed microarrays.

We describe the fabrication of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures with a high aspect ratio and the use of hydrogel microstructures containing the enzyme beta-galactosidase (beta-Gal) or glucose oxidase (GOx)/horseradish peroxidase (HRP) as biosensing components for the simultaneous detection of multiple analytes. The diameters of the hydrogel microstructures were almost the same at the top and at the bottom, indicating that no differential curing occurred through the thickness of the hydrogel microstructure. Using the hydrogel microstructures as microreactors, beta-Gal or GOx/HRP was trapped in the hydrogel array, and the time-dependent fluorescence intensities of the hydrogel array were investigated to determine the dynamic uptake of substrates into the PEG-DA hydrogel. The time required to reach steady-state fluorescence by glucose diffusing into the hydrogel and its enzymatic reactions with GOx and HRP was half the time required for resorufin beta-D-galactopyranoside (RGB) when used as the substrate for beta-Gal. Spatially addressed hydrogel microarrays containing different enzymes were micropatterned for the simultaneous detection of multiple analytes, and glucose and RGB solutions were incubated as substrates. These results indicate that there was no cross-talk between the beta-Gal-immobilizing hydrogel micropatches and the GOx/HRP-immobilizing micropatches.

Quantitative analysis of methyl parathion pesticides in a polydimethylsiloxane microfluidic channel using confocal surface-enhanced Raman spectroscopy.

A fast and ultra-sensitive trace analysis of methyl parathion pesticides in a polydimethylsiloxane (PDMS) microfluidic channel was investigated using confocal surface-enhanced Raman spectroscopy (SERS). A three-dimensional PDMS-based passive micromixer was fabricated for this purpose. This PDMS micromixer showed a high mixing efficiency because a strong chaotic advection was developed by the simultaneous vertical and transverse dispersion of the confluent streams. The confocal SERS signal was measured after methyl parathion pesticides were effectively adsorbed onto silver nanoparticles while flowing along the upper and lower alligator-teeth-shaped PDMS channel. A quantitative analysis of the methyl parathion pesticides was performed based on the measured peak height at 1246 cm-1. Our method has a detection limit of 0.1 ppm. This value satisfies the requirement recommended by the Collaborative International Pesticides Analytical Council (CIPAC) for the determination of methyl parathion in pesticide formulations. This study demonstrates the feasibility of using confocal SERS for the highly sensitive detection of methyl parathion pesticides in a PDMS microfluidic channel.

Highly sensitive signal detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip: confocal surface-enhanced Raman spectroscopic study.

Rapid and highly sensitive detection of duplex dye-labelled DNA sequences in a PDMS microfluidic channel was investigated using confocal surface enhanced Raman spectroscopy (SERS). This method does not need either an immobilization procedure or a PCR amplification procedure, which are essential for a DNA microarray chip. Furthermore, Raman peaks of each dye-labelled DNA can be easily resolved since they are much narrower than the corresponding broad fluorescence bands. To find the potential applicability of confocal SERS for sensitive bio-detection in a microfluidic channel, the mixture of two different dye-labelled (TAMRA and Cy3) sex determining Y genes, SRY and SPGY1, was adsorbed on silver colloids in the alligator teeth-shaped PDMS microfluidic channel and its SERS signals were measured under flowing conditions. Its major SERS peaks were observable down to the concentration of 10(-11) M. In the present study, we explore the feasibility of confocal SERS for the highly sensitive detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip.

Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry.

Aminophenylboronic acid (APBA) has been immobilized on magnetic beads for the direct determination of glycoprotein by matrix assisted laser desorption/ionizaton time of flight mass spectrometry (MALDI-TOF-MS). An APBA layer was formed on the surface of carboxylic acid terminated magnetic beads by coupling with carbodiimide and subsequently reacted with an N-hydroxysuccinimide moiety. The immobilized APBA was identified by MALDI-TOF-MS without a matrix. Glycoproteins, such as HbA1c, fibrinogen, or RNase B were separated and desalted using APBA magnetic beads by simply washing the magnetic beads and then separating them by external magnet. Proteins can be identified by direct determination of proteins on beads on MALDI plate and confirmed again by peptide mass finger printing after digestion of proteins on magnetic beads by trypsin. Fluorescence image with a FITC tagging protein using confocal laser microscopy showed the difference of immobilization efficiency between glycoproteins and nonglycoproteins. The methods developed within this work allow the simple treatment and enrichment of glycoproteins as well as direct determination of proteins on beads by MALDI-TOF-MS.

Analysis of protein adsorption characteristics to nano-pore silica particles by using confocal laser scanning microscopy.

The effect of average pore size of nano-pore silica particles on protein adsorption characteristics was determined experimentally by the dissociation constant and the adsorption capacity determined from the Langmuir equation. As the average pore size was increased from 2.2 to 45 nm, the BSA adsorption capacity increased from 16.8 to 84.3 mg/g-silica so as the equilibrium constant (from 2.6 to 9.4 mg/ml). Using confocal microscopy with fluorescence labeling, we could visualize the protein adsorption in situ and determine the minimum pore size required for efficient intraparticle adsorption. The confocal microscopy analysis revealed that BSA was adsorbed mainly on the surface of the particles with a smaller pore size, but diffused further into the interstitial surface when it was sufficiently large. It was concluded that for BSA whose Stoke's diameter is ca. 3.55 nm the minimum pore size of about 45 nm or larger was required for a sufficient adsorption capacity.

Analysis of passive mixing behavior in a poly(dimethylsiloxane) microfluidic channel using confocal fluorescence and Raman microscopy.

Confocal fluorescence microscopy (CFM) and confocal Raman microscopy (CRM) have been applied to monitor the laminar flow mixing behavior in a poly(dimethylsiloxane) (PDMS) microfluidic channel. Two passive PDMS micromixing devices were fabricated for this purpose: a two-dimensional round-wave channel and a three-dimensional serpentine channel. The microscale laminar flow mixing of ethanol and isopropanol was evaluated using the CFM and CRM at various flow rates. The mixing behavior of confluent streams in the microchannel was assessed by determining the degree of color change in Rhodamine 6G dye on mixing using the CFM. However, it was also possible to quantitatively evaluate the mixing process without employing a fluorescence label using the CRM. The results show a strong potential for CRM as a highly sensitive detection tool to measure fundamental fluid mixing processes and to provide detailed information on chemical changes of non-fluorescent reaction mixtures in a PDMS microfluidic channel.

Enhanced delivery of T cells to tumor after chemotherapy using membrane-anchored, apoptosis-targeted peptide.

Chemotherapy-induced apoptosis of tumor cells enhances the antigen presentation and sensitizes tumor cells to T cell-mediated cytotoxicity. Here we harnessed the apoptosis of tumor cells as a homing signal for the delivery of T cells to tumor. Jurkat T cells were anchored with ApoPep-1, an apoptosis-targeted peptide ligand, using the biocompatible anchor for membrane (BAM), an oleyl acid derivative. The ApoPep-1-BAM conjugate was efficiently anchored to cell membrane, while little anchoring was obtained with ApoPep-1 alone. The retention period of the ApoPep-1-BAM conjugate on cell membrane was approximately 80 and 40 min in the absence and presence of serum, respectively. ApoPep-1 was resistant to degradation in serum until 2h. The apoptosis-targeted T cells that were anchored with the ApoPep-1-BAM preferentially bound to apoptotic tumor cells over living cells. When intravenously injected into tumor-bearing mice, the number of apoptosis-targeted T cells and in vivo fluorescence signals by the homing of the cells to doxorubicin-treated tumor were higher than those of untargeted T cells. Accumulation of apoptosis-targeted T cells at other organs such as liver was not detected. These results suggest that the chemotherapy-induced apoptosis and subsequent enhancement of T cell delivery to tumor by the membrane anchoring of the apoptosis-targeted peptide could be a novel strategy for cancer immunotherapy.

Fabrication of porous extracellular matrix scaffolds from human adipose tissue.

Adipose tissue is found over the whole body and easily obtained in large quantities with minimal risk by a common surgical operation, liposuction. Although liposuction was originally intended for the removal of undesired adipose tissue, it may provide an ideal material for tissue engineering scaffolds. Here we present novel, porous scaffolds prepared from human adipose tissues. The scaffolds were fabricated in a variety of macroscopic shapes such as round dishes, squares, hollow tubes, and beads. The microscopic inner porous structure was controlled by the freezing temperature, with a decrease in pore size as the freezing temperature decreased. The scaffold prepared from human adipose tissue contains extracellular matrix components including collagen. Preliminary in vitro studies showed that human adipose-derived stem cells attached to a human extracellular matrix scaffold and proliferated. This scaffold based on human adipose tissue holds great promise for many clinical applications in regenerative medicine, particularly in patients requiring soft-tissue regeneration.

Preparation of monodisperse and size-controlled poly(ethylene glycol) hydrogel nanoparticles using liposome templates.

Liposomes were used as templates to prepare size-controlled and monodisperse poly(ethylene glycol) (PEG) hydrogel nanoparticles. The procedure for the preparation of PEG nanoparticles using liposomes consists of encapsulation of photopolymerizable PEG hydrogel solution into the cavity of the liposomes, extrusion through a membrane with a specific pore size, and photopolymerization of the contents inside the liposomes by UV irradiation. The size distributions of the prepared particles were 1.32+/-0.16 microm (12%), 450+/-62 nm (14%), and 94+/-12 nm (13%) after extrusion through membrane filters with pore sizes of 1 microm, 400 nm, and 100 nm, respectively. With this approach, it is also possible to modify the surface of the hydrogel nanoparticles with various functional groups in a one-step procedure. To functionalize the surface of a PEG nanoparticle, methoxy poly(ethylene glycol)-aldehyde was added as copolymer to the hydrogel-forming components and aldehyde-functionalized PEG nanoparticles could be obtained easily by UV-induced photopolymerization, following conjugation with poly-L-lysine-FITC through amine-aldehyde coupling. The prepared PEG particles showed strong fluorescence from FITC on the edge of the particles using confocal microscopy. The immobilization of biomaterials such as enzymes in hydrogel particles could be performed with loading beta-galactosidases during the hydration step for liposome preparation without additional procedures. The resorufin produced by applying resorufin beta-D-galactopyranoside as the substrate showed the fluorescence under the confocal microscopy.

Ultra-sensitive trace analysis of cyanide water pollutant in a PDMS microfluidic channel using surface-enhanced Raman spectroscopy.

Rapid and highly sensitive trace analysis of cyanide water pollutant in an alligator teeth-shaped PDMS microfluidic channel was investigated using surface-enhanced Raman spectroscopy. Compared with previously reported analytical methods, the detection sensitivity was enhanced by several orders of magnitude.

Continuous fabrication of biocatalyst immobilized microparticles using photopolymerization and immiscible liquids in microfluidic systems.

We report a novel technique for manufacturing polymeric microparticles containing biocatalysts by the behavior of immiscible liquids in microfluidic systems and in situ photopolymerization. The approach utilizes a UV-polymerizable hydrogel/enzyme solution and an immiscible oil solution. The oil and hydrogel solutions form emulsions in pressure-driven flow in microchannels at high values of the dimensionless capillary number (Ca). The resultant hydrogel droplets are then polymerized in situ via exposure to 365 nm UV light. This technique allows for the generation of monodisperse particles whose size can be controlled by the regulation of flow rates. In addition, both manufacturing microparticles and immobilizing biocatalysts can be performed simultaneously and continuously.

Enzymic cleavage of fusion protein using immobilized urokinase covalently conjugated to glyoxyl-agarose.

We immobilized urokinase (UK) by covalent attachment to activated Sepharose 6B-CL through multi-point amine coupling and evaluated its performance in cleaving a fusion protein, which consisted of recombinant human growth hormone (hGH) and a fragment of glutathione S-transferase that was linked by a tetrapeptide of a UK-specific recognition sequence. Packing densities of aldehyde groups on the activated agarose surface could be controlled in a gel range of 7-60 micromol/ml aldehyde by the amount of glycidol used. The immobilization yield was nearly 100% at pH 10.5, and the specific activity of the immobilized UK was equivalent to about 80% of soluble UK under the assay conditions. The immobilized UK showed an improvement in pH and thermal stability, probably due to the structural rigidity imparted by multi-point linkages to the matrix. The cleavage rate by the immobilized UK was lower than that of the soluble enzyme but the side reaction of cryptic cleavage was significantly decreased, which might suggest that the enzyme's specificity was altered by the immobilization. Cleavage yield in the column packed with immobilized UK was dependent on the feed rate, and the yield was approx. 80% of that of the soluble UK. The monomeric hGH could be obtained by selectively precipitating the uncleaved fusion protein and the GST fragments at an acidic pH.