Nanoporous alumina membranes were utilized as cell culture substrates. Two different anodization processes were applied in order to obtain highly ordered, self-supporting nanoporous alumina membranes. The resulting membranes exhibit uniform nanopores with constant diameters having aspect ratios (pore depth:pore diameter) larger than 1000. Tentative experiments evaluating the cellular interaction of a hepatoma cell line (HepG2) with the nanoporous alumina membranes will be described. Furthermore, cell proliferation as well as the adhesion behavior of the cells on the porous substrates was investigated. Scanning electron microscopy (SEM) and focused ion beam (FIB) technology were applied to examine the cell morphology. The experiments revealed that the nanoporous membranes can be used as cell culture substrates and that they have no adverse effects on cell proliferation and cell viability. Besides, cells on membranes with pore diameters larger than 200 nm developed small cell extensions (filopodia) which penetrated into the nanopores.

Nanoparticles are widely applied as diagnostic reagents, drug-delivery carriers or bioaffinity beads. In particular, magnetic particles have been widely used in the study of biomedical applications. When we look ahead to the future, it is needed to construct precisely designed surface containing such as high bioaffinity and capability to capture of small amount protein or specific protein among various protein. As a method to construct precisely designed biocompatible polymer surface, atom transfer radical polymerization (ATRP) was widely used as a living free radical polymerization. ATRP has advantages such as control of molecular weight, narrow polydispersivity, easy synthesis of block copolymer, and preparation of high density polymer brush under mild condition. Previously, we have demonstrated synthesis of poly (2-methacryloyloxyethyl phosphorylcholine (MPC)) brush on ferric oxide (Fe3O4) by ATRP from immobilized initiator includes bromine that ATRP moiety. Here, it is known that poly(MPC) has been not affected for reduction of nonspecific binding, salt concentrations, pH and temperature. In this state, the poly(MPC)-grafted Fe3O4 could not be used for diagnostic material and collection material using magnetic interaction because poly(MPC) suppress protein adsorption. To introduce immobilization part for protein, p-nitrophenyloxycarbonyl polyethylene glycol methacrylate (MEONP) monomer that has active ester was polymerized as a block component. The poly(MPC-b-MEONP)-grafted Fe3O4 could capture proteins via active ester to amine of proteins. However, on careful consideration, this immobilization method had disadvantage for capture protein because immobilization part located in the side chain, not locate most outer surface. We focused on bromine (Br) located on end group of poly(MPC) brush, probably locate most outer surface. If the end group would be modified easily, the technique will be most effective modification. We adapted the reduction from azide (N3) to amine (NH2), after Br was exchanged to N3. This reduction has been called “staudinger reduction”. Generally, Click chemistry that reaction between N3 and alkyne is famous as a reaction of N3. On the other hand, the modification technique has wide application range due to amine group. At first, bromine was converted into azide, N3 using NaN3. Furthermore, the N3 was reduced to NH2 using PPh3 and H2O. The purification of magnetic nanoparticles with end group modified poly(MPC) brush was easy because of use of external magnetic interaction. The surface characterization at each step was estimated by XPS and FT-IR spectrum measurement. The absorbance signal attributed to N3 was appeared around 2000 cm-1 from FT-IR spectrum and N1s signal according to NH2 was observed at 399 eV from XPS measurement. The surface NH2 could react to carboxyl group or convert into other functional groups easily. The end group modification will be a useful for various bioaffinity bead applications.

We present the design of two biointerfaces on a SiO2 substrate for single stranded DNA (ssDNA) immobilization using either covalent grafting or electrostatic adsorption. The influence of the type of biointerface on the rate of diffusion-limited hybridization reaction with complementary ssDNA from a solution is studied. Patterning of the biointerfacefunctionalization layers and the scaling down of the reaction volumes to µL range is demonstrated. The use of externally applied electric field pulses is shown to accelerate the hybridization reaction kinetics to the sub-ms time scale.

Well established quartz crystal microbalance with dissipation (QCM-D) monitoring technique has been applied here for investigation the adsorption kinetics of three different proteins (BSA, IgG, Lysozyme) on thin film of liquid crystal copper octakishexylthiophthalocyanine [(C6S)8PcCu] surface. Modification of phthalocyanine surface by protein coating can be useful for photo dynamic therapy or in biomedical devices purpose in order to increase biocompatibility. The adsorption kinetics on vertically oriented molecular stacks of phthalocyanines were different for three proteins. Lysozyme reached to saturation quickly compare to BSA and IgG. Changes in resonance frequency and dissipation for three overtones were fitted with the Voigt viscoelastic model to calculate the shear viscosity and shear elastic modulus for the adsorbed proteins. Sauerbrey equation was used to calculate approximate adsorbed mass directly from frequency change. Adsorbed mass calculated from Voigt model was different from Sauerbrey equation. Hydration level of adsorbed protein layer was calculated from the difference in mass based on above two methods which was increased with concentration from 10-80% for BSA and IgG whereas it decreased from 20-1% for Lysozyme. Variation of viscosity and shear elastic modulus with concentration is opposite for BSA, IgG than Lysozyme. Depending on the hydration levels, adsorbed BSA and IgG formed soft (viscoelastic) layer compare to rigid layer of Lysozyme on (C6S)8PcCu surface.

Preparation of nonbiofouling surface and micropatterned biorecognition layer over nonbiofouling for highly sensitive microarray biosensors, was conducted by constructing biocompatible poly(2-methacryloyloxyethyl phosphorylcholine(MPC)) brushes on polymer substrates based on living radical polymerization using macrophotoiniferters comprised of 2-ethylhexyl methacrylate (EHMA) and 4-vinylbenzyl N,N-diethyldithiocarbamate (VBDC). Protein adsorption on these modified surfaces significantly reduces compared to on polymeric substrate and the micropatterning consisted of poly(MPC) brush and biorecognition layer can be easily done by applying photomask with high fidelity.