Ji-Hye Lee

Preparation of orthogonally functionalized surface using micromolding in capillaries technique for the control of cellular adhesion

This study presents a simple method for the fabrication of an orthogonal surface that can be applied for cell patterning without the need to immobilize specific adhesive peptides, proteins, or extracellular matrix (ECM) for cell attachment. Micromolding in capillaries (MIMIC) produced two distinctive regions. One region contained poly(ethylene glycol)-poly(D,L-lactide) diblock copolymer (PEG-PLA) designed to provide a biological barrier to the nonspecific binding of proteins and fibroblast cells. The other region was coated with polyelectrolyte (PEL) to promote the adhesion of biomolecules including proteins and cells. Resistance to the adsorption of proteins increased with the length of PEG and PLA chains because the longer PEG chain increased the PEG layer thickness and the longer PLA chain induced stronger interaction with the PEL surface. The PEG5k-PLA2.5k (20mg/ml) was the most efficient candidate for the prevention of protein adhesion among the PEG-PLA copolymers examined. The orthogonal functionality of prepared surfaces having PEL regions and background PEG-PLA regions resulted in rapid patterning of biomolecules. Fluorescein isothiocyanate-tagged bovine serum albumin (FITC-BSA) and fibroblast cells successfully adhered to the exposed PEL surfaces. Although methods for cell patterning generally require an adhesive protein layer on the desired area, these fabricated surfaces without adhesive proteins provide a gentle microenvironment for cells. In addition, our proposed approach could easily control patterns, sizes, and shapes at micron scale.

Laser desorption/ionization—Mass spectrometry using mesoporous silicate as matrix for the analysis of various molecules

In this study, mesoporous silicate was applied as a matrix for the analysis of various molecules from small molecules to medium sized peptides in laser desorption/ionization mass spectrometry. In contrast with conventional matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), the proposed approach desorption/ionization on mesoporous silicate mass spectrometry (DIOM-MS), significantly reduces the problem of matrix interference in low mass region and can be applied to the analysis of versatile chemicals including amino acids, synthetic drugs, peptides and others. In addition, distinctive advantage of DIOM-MS showed higher salt tolerance and could be applied to identify the proteins from the analysis of tryptically digested peptides. DIOM-MS has several availabilities such as easy sample preparation, rapid analysis of small molecules without noise, peptide analysis without organic matrix, high salt tolerance, versatile coupling with other separation techniques, and high throughput manner.

Generation of uniform agarose microwells for cell patterning by micromolding in capillaries

AbstractWe described a simple and facile method to generate uniform agarose microwells on a polyelectrolyte (PEL) multilayer functionalized surface for efficient and reliable cell patterning. The PEL multilayers, composed of polyallylamine hydrochloride (PAH) and polystyrene sulfonate ammonium (PSS), provide an adhesive environment, which promotes cell proliferation in live cell-based assays. Agarose microwells, which are able to selectively isolate cells into individual compartments, are fabricated by micromolding the agarose solution in capillaries (MIMIC). Moreover, the fabricated agarose microwells are able to effectively form different shapes (e.g., circles, triangles, squares, and stars) and can isolate multiple cell types (e.g., HEK 293, NIH3T3, and HepG2). We also demonstrated how this technique can be used for the real-time monitoring of communication between primary neuronal cells in the agarose microwells.

Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry : A tool for functional genomics

For the rapid identification of functional activity of unknown genes from a sequence database, a new method based on in vitro protein synthesis combined with mass spectrometry was developed. To discriminate their subtle enzymatic activity, in vitro synthesized and one-step purified lipolytic enzymes, such as lip A and lip B from Bacillus subtilis and an unknown protein ybfF from Escherichia coli, were reacted with a mixture of triglycerides with different carbon chain lengths. Using direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of reaction product, all three enzymes were revealed to have strong esterase activity rather than true lipase activity, which has no reactivity on long-chain fatty acids such as triolein. These results were also confirmed by classical color assay using p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as representative lipolytic substrates.

Encapsulation of cell into monodispersed hydrogels on microfluidic device

In here, we present the microfluidic approach to produce monodispersed microbeads that will contain viable cells. The utilization of microfludics is helpful to synthesize monodispersed alginate hydrogels and in situ encapsulate cell into the generating hydrogels in microfludic device. First, the condition of formation of hydrogels in multiphase flows including oil, CaCl2, and alginate was optimized. Based on the preliminary survey, microfludic device could easily manipulate the size of alginate beads having narrow size distribution. The microfluidic method manipulates the size of hydrogel microbeads from 30 to 200um with a variation less than 2%. For the proof of concept of cell entrapment, the live yeast expressing green fluorescence protein is successfully encapsulated in microfluidic device.

Simple micropatterning of proteins using polyelectrolyte multilayers and microcontact printing

The selective immobilization of various biomolecules in well-defined area is important technique for the development of biosensors and biochips. Especially, the fabrication of protein micropatterns preserving their functional activity on the desired surface is critical issue for the development of medical diagnostic devices and basic protein studies. In this study, we have introduced a simple but reliable method of protein patterning on functionalized polyelectrolyte thin films (PEL) through consecutive layer-by-layer adsorption of polyelectrolytes via self-assembly technique and microcontact printing (μCP). For the selection of appropriate surface, several representative surfaces modified with various functional materials including aldehyde, epoxide, poly-L-lysine, amine, and self-assembled polyelectrolyte multilayers (PEL) were investigated. The PEL surface providing electrostatic interaction force showed most high functionality in point of homogeneous patterning of proteins with high density and preservation of inherent 3-dimensional structure of proteins. Immunoassay as a model system of protein-protein interaction showed good linearity, indicating the feasibility of a quantitative measurement of the concentration of target proteins in sample. Our proposed approach based on PEL constructed by self-assembly technique in aqueous solution is green chemistry and cost-effective method to generate stable 3-D thin film on surface. The demand for strict control over the positioning and the stable immobilization of several kinds of biomolecules in fabricated structures can result in many applications.

Rapid bio-patterning method based on the fabrication of PEG microstructures and layer-by-layer polymeric thin film

The patterning of biomolecules in well-defined microstructures is critical issue for the development of biosensors and biochips. However, the fabrication of microstructures with well-ordered and spatially discrete forms to provide the patterned surface for the immobilization of biomolecules is difficult because of the lack of distinct physical and chemical barriers separating patterns. This study present rapid biomolecule patterning using micromolding in capillaries (MIMIC), soft-lithographic fabrication of PEG microstructures for prevention of nonspecific binding as a biological barrier, and self assembled polymeric thin film for efficient immobilization of proteins or cells. For the proof of concept, protein (FITC-BSA), bacteria (E.coli BL21-pET23b-GFP) were used for biomolecules patterning on polyelectrolyte coated surface within PEG microstructures. The novel approach of MIMIC combined with LbL coating provides a general platform for patterning a broad range of materials because it can be easily applied to various substrates such as glass, silicon, silicon dioxide, and polymers.