Hyuk Ko

Development of SPR biosensor for the detection of human hepatitis B virus using plasma-treated parylene-N film

A plasma-treated parylene-N film was presented for the immobilization of proteins through physical adsorption. The changes in surface properties of the parylene-N film after plasma-treatment were analyzed using contact angle microscopy and AFM. To demonstrate the high protein-immobilization efficiency of the plasma-treated parylene-N film, the immobilization efficiencies of differently modified surfaces were compared using model proteins with different surface charges, such as streptavidin (pI=5, negatively charged at pH 7), horseradish peroxidase (pI=6.6, nearly neutral at pH 7), and avidin (pI=10, positively charged at pH 7). The application of the plasma-treated parylene-N film as an SPR biosensor was also tested by immobilizing model proteins. An SPR biosensor based on the plasma-treated parylene-N film was developed for the detection of the human hepatitis virus surface antigen (HBsAg), and the plasma-treated parylene-N film was estimated to improve the sensitivity of SPR biosensor as much as 1000-fold by enhancing immobilization of receptor antibodies.

One step immobilization of peptides and proteins by using modified parylene with formyl groups.

One-step immobilization method for peptides and proteins is developed by using modified parylene film with formyl groups which is suitable for microplate-based immunoassay and SPR biosensor application. The immobilization of peptides and proteins is achieved through the covalent bonding of the formyl group with the primary amine groups of peptides and proteins, which no additional activation step is required. In this work, the immobilization efficiency of parylene-H is estimated in comparison with parylene-A and physical adsorption, using biotinylated-cyclic citrullinated peptide (biotinylated-CCP), human chorionic gonadotropin (hCG) and horseradish peroxidase (HRP) as model proteins. The applicability of parylene-H film to SPR biosensor is demonstrated by estimating the detection range and sensitivity of SPR biosensor at various thicknesses. The immobilization efficiency of parylene-H film for SPR biosensor was compared with physical adsorption by using HRP as a model protein.

Covalent protein immobilization with a parylene‐H film for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

RATIONALE For the sensitive analysis of receptor-ligand interactions by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), receptor proteins should be immobilized on a target plate with a high surface density. In this work, a parylene-H film with formyl groups was developed for the efficient covalent immobilization of receptor proteins for MALDI-TOF MS. METHODS The parylene-H film was thermally deposited on a target plate and receptor proteins were covalently immobilized. The surface properties of the parylene-H film were analyzed by atomic force microscopy (AFM) and cyclic voltammetry (CV). The immobilization efficiency of the parylene-H film was analyzed by fluorescence imaging with streptavidin and fluorescence-labeled biotin. MALDI-TOF MS was performed using the parylene-H-coated target plate with streptavidin and different concentrations of biotinylated peptide as the receptor and ligand, respectively. RESULTS The parylene-H film on a target plate had a flat surface (Rq : ±2.755 nm) without any pinholes and could be regarded to be electrically conductive under an electric potential of 30 kV. The fluorescence image proved that the parylene-H film improved the protein immobilization efficiency as well as ligand detection sensitivity. The mass spectra quantitatively revealed peaks from the ligand molecules without any interference peaks from the immobilized receptor proteins. CONCLUSIONS A parylene-H film with formyl groups was thermally deposited on a target plate and the receptor protein was covalently immobilized on the target plate. The interactions of ligand molecules with the immobilized receptor proteins were quantitatively analyzed by MALDI-TOF MS.

UV-irradiated parylene surfaces for proliferation and differentiation of PC-12 cells

PC-12 cells originate from neuroblastic cells, which have an ability to differentiate into neuronlike cells. In this work, the purpose was to estimate the influence of microenvironments on cell attachment and neuritogenesis capacity of PC-12 cells on parylene-N and parylene-C films with and without ultraviolet (UV) light treatment. The estimate of total cell number after incubation for 72h, the ratio of adherent to suspended cells, counting of neurite outgrowths on parylene-N or parylene-C films after UV exposure suggested that these films were suitable for proliferation as well as differentiation of PC-12 cells. The differences in surface properties of parylene-N and parylene-C films with and without UV exposure were analyzed by contact angle measurement, Fourier-transform infrared spectrometry, and X-ray photoelectron spectroscopy. According to these analyses, introduction of oxygen-related chemical functional groups was presumed to result in increased hydrophilicity and efficiency of protein immobilization on parylene-N and parylene-C films after UV treatment. According to fluorescent staining, western blotting, and cell cycle analysis, UV-treated parylene-C and parylene-N films appear to effectively facilitate simultaneous proliferation and differentiation of PC-12 cells with neurite outgrowth.