Tatsuro Goda

Super-hydrophilic silicone hydrogels with interpenetrating poly(2-methacryloyloxyethyl phosphorylcholine) networks.

We synthesized silicone hydrogels from 2-methacryloyloxyethyl phosphorylcholine (MPC) and bis(trimethylsilyloxy)methylsilylpropyl glycerol methacrylate (SiMA) using two methods: random copolymerization with a small amount of cross-linker (P(SiMA-co-MPC)) and construction of an interpenetration network (IPN) structure composed of cross-linked poly(MPC)(PMPC) chains and cross-linked poly(SiMA)(PSiMA) chains (PSiMA-ipn-PMPC). The polymerization was carried out by photoreaction. The surface hydrophilicity and water absorbability of P(SiMA-co-MPC) increased with an increase in the MPC unit composition. On the other hand, in the case of PSiMA-ipn-PMPC, a super-hydrophilic surface was obtained by the surface enrichment of MPC units. The optical and mechanical properties of PSiMA-ipn-PMPC are suitable for use as a material for preparing contact lenses. In addition, the oxygen permeability of PSiMA-ipn-PMPC remains high because of the PSiMA chains. The MPC units at the surface of the hydrogels reduce protein adsorption effectively. From these results for PSiMA-ipn-PMPC, we confirmed that it has the potential for application to silicone hydrogel contact lenses.

Cell-penetrating macromolecules: Direct penetration of amphipathic phospholipid polymers across plasma membrane of living cells

Nanoscaled materials are normally engulfed in endosomes by energy-dependent endocytosis and fail to access the cytosolic cell machinery. Although some biomolecules may penetrate non-endocytically or fuse with plasma membranes without overt membrane disruption, to date no synthetic macromolecule of comparable size has been shown to exhibit this property. Here, we discovered mechanism of direct cell membrane penetration using synthetic phospholipid polymers. These water-soluble amphiphilic phospholipid polymers enter the cytoplasm of living mammalian cells in vitro within a few minutes without overt bilayer disruption even under conditions where energy-dependent endocytic uptakes are blocked. Furthermore, targeted cytosolic distribution to cell organelles was achieved by selecting specific fluorescent tags to the polymers. Thus, the phospholipid polymers can provide a new way of thinking about access to the cellular interior, namely direct membrane penetration.

Protein adsorption resistance and oxygen permeability of chemically crosslinked phospholipid polymer hydrogel for ophthalmologic biomaterials

The biomimetic structure of a polymer hydrogel bearing phosphorylcholine groups was obtained from 2-methacryloyloxyethylphosphorylcholline (MPC) and a novel crosslinker, 2-(methacryloyloxy)ethyl-N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), to prepare biocompatible ocular materials. MMPC is a dimethacrylate with phosphorylcholine-analogous linkage. Previous reports clarified that the affinity of MMPC to MPC enables the water contents and mechanical properties of the poly(MPC) hydrogels to be varied without disturbing the bulk phases. In this study, we examined the protein adsorption resistance, water wettability, oxygen permeability, and electrolyte permeability of the mechanically enhanced poly(MPC) hydrogel crosslinked with MMPC. The amount of protein adsorbed on this hydrogel was 0.9 microg/cm(2), which accounted for 30% of Omafilcon A and 3% of Etafilcon A. Water contact angle experiments revealed the high wettability of the poly(MPC) hydrogels. The oxygen permeability and NaCl diffusion constant of the poly(MPC) hydrogels were 64 barrer and 48 x 10(-6) cm(2)/s, respectively. This high permeability resulted from the high water content, similar to the case of the human cornea. These results suggested that poly(MPC) hydrogels have good potential for use in ophthalmologic biomaterials.

Label-free and reagent-less protein biosensing using aptamer-modified extended-gate field-effect transistors.

We have developed biosensors based on an aptamer-modified field-effect transistor (FET) for the detection of lysozyme and thrombin. An oligonucleotide aptamer as a sensitive and specific ligand for these model proteins was covalently immobilized on a gold electrode extended to the gate of FET together with thiol molecules to make a densely packed self-assembled monolayer (SAM). The aptamer-based potentiometry was achieved in a multi-parallel way using a microelectrodes array format of the gate electrode. A change in the gate potential was monitored in real-time after introduction of a target protein at various concentrations to the functionalized electrodes in a buffer solution. Specific protein binding altered the charge density at the gate/solution interface, i.e., interface potential, because of the intrinsic local net-charges of the captured protein. The potentiometry successfully determined the lysozyme and thrombin on the solid phase with their dynamic ranges 15.2-1040 nM and 13.4-1300 nM and the limit of detection of 12.0 nM and 6.7 nM, respectively. Importantly, robust signals were obtained by the specific protein recognition even in the spiked 10% fetal bovine serum (FBS) conditions. The technique herein described is all within a complementary metal oxide semiconductor (CMOS) compatible format, and is thus promising for highly efficient and low cost manufacturing with the readiness of downsizing and integration by virtue of advanced semiconductor processing technologies.

Photografting of 2-methacryloyloxyethyl phosphorylcholine from polydimethylsiloxane: Tunable protein repellency and lubrication property

The phosphorylcholine group functional methacrylate monomer, 2-methacryloyloxyethyl phosphorylcholine (MPC), was graft polymerized from the polydimethylsiloxane (PDMS) substrate using ultraviolet irradiation and using benzophenone as a photoinitiator. The varying monomer concentrations and irradiation times were investigated in order to verify the relationships between graft density and protein resistance under specific biological conditions. The ellipsometry analysis revealed that the layer thickness of the grafted polymer depended on the monomer concentrations after the irradiation for 1 min, however, it stabilized thereafter in all the specified conditions. The curve fitting of the C1s spectrum obtained by X-ray photoelectron spectroscopy analysis showed that the amount of grafted polymer increased with an increase in both monomer concentration and irradiation time. Atomic force microscopic images revealed that the terminations among the graft chains became dominant due to magnified chain mobility followed by growth of their length. In vitro albumin and fibrinogen adsorption results indicated that the resistance to protein adsorption was easily tuned by the specified conditions due to the controlled graft density. Lubrication was dramatically enhanced by the grafting and it was further promoted by an increase in the graft density in good solvents, indicating that the interactions between the graft chains and the solvents resulted in the lubrication system. These basic findings regarding the grafted PDMS surface are important for versatile applications, including its use as a biomaterial and microfluidic device.

Thiolated 2-methacryloyloxyethyl phosphorylcholine for an antifouling biosensor platform

We developed a new building block for a protein- and cell-repellant self-assembled monolayer (SAM) from 2-methacryloyloxyethyl phosphorylcholine (MPC) via a simple Michael-type addition to one mercapto group in alkanedithiol. The thiolated MPC can enable functionalization of a noble metal electrode to minimize noise signal in biosensing.

Interpretation of protein adsorption through its intrinsic electric charges: a comparative study using a field-effect transistor, surface plasmon resonance, and quartz crystal microbalance.

We describe the highly sensitive detection of the nonspecific adsorption of proteins onto a 1-undecanethiol self-assembled monolayer (SAM)-formed gold electrode by parallel analysis using field effect transistor (FET), surface plasmon resonance (SPR), and quartz crystal microbalance (QCM) sensors. The FET sensor detects the innate electric charges of the adsorbed protein at the electrode/solution interface, transforming the change in charge density into a potentiometric signal in real time, without the requirement for labels. In particular, using the Debye-Huckel model, the degree of potential shift was proportional to the dry mass of adsorbed albumin and β-casein. A comparison of the FET signal with SPR and QCM data provided information on the conformation and orientation of the surface-bound protein by observing characteristic break points in the correlation slopes between the signals. These slope transitions reflect a multistage process that occurs upon protein adsorption as a function of protein concentration, including interim coverage, film dehydration, and monolayer condensation. The FET biosensor, in combination with SPR and QCM, represents a new technology for interrogating protein-material interactions both quantitatively and qualitatively.

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

Detection of Microenvironmental Changes Induced by Protein Adsorption onto Self-Assembled Monolayers using an Extended Gate-Field Effect Transistor

Nonspecific protein adsorption on self-assembled monolayer (SAM) alkanethiols with various terminal groups was investigated qualitatively and quantitatively using an extended gate-field effect transistor (extended gate-FET). The SAMs were characterized by XPS, cyclic voltammogram and water contact angle measurements. Changes in gate voltage of 1 mV caused by intrinsic charges of adsorbed protein on an undecanethiol SAM in 15 mM Dulbeccos phosphate buffered saline were equivalent to 3.6 ng cm(-2), 1.3 ng cm(-2), and 16 ng cm(-2) for bovine serum albumin (BSA), lysozyme, and bovine plasma fibrinogen (BPF), respectively, as calculated by the Debye-Huckel model. Adsorption coefficients, maximum adsorption densities, and Gibbs free energies of adsorption were successfully determined using the Langmuir equation. The isotherms depended on the surface properties of the SAMs for BSA and lysozyme adsorption. In contrast, changes in gate voltage were almost independent of SAM type for BPF adsorption. Adsorption of large proteins may not be quantitatively detected because of the large dimensions of the biomolecules compared with the Debye length. In summary, the FET measurement is a nonlabeling, highly sensitive, and quantitative method for detecting nonspecific adsorption of small proteins with dimensions that are comparable to the Debye length of a solution.

A label-free electrical detection of exosomal microRNAs using microelectrode array

We report a method for detecting microRNAs encapsulated in exosomes using a microelectrode array in semiconductor-based potentiometry after RT-PCR. The inherent miniaturization of the electrical biosensor meets requirements for massively parallel analysis of circulating microRNA as a noninvasive biomarker.