Inseong Choi

Preparation of Gradient Surfaces by Using a Simple Chemical Reaction and Investigation of Cell Adhesion on a Two‐Component Gradient

This article describes a simple method for the generation of multicomponent gradient surfaces on self-assembled monolayers (SAMs) on gold in a precise and predictable manner, by harnessing a chemical reaction on the monolayer, and their applications. A quinone derivative on a monolayer was converted to an amine through spontaneous intramolecular cyclization following first-order reaction kinetics. An amine gradient on the surface on a scale of centimeters was realized by modulating the exposure time of the quinone-presenting monolayer to the chemical reagent. The resulting amine was used as a chemical handle to attach various molecules to the monolayer with formation of multicomponent gradient surfaces. The effectiveness of this strategy was verified by cyclic voltammetry (CV), matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), MS imaging, and contact-angle measurements. As a practical application, cell adhesion was investigated on RGD/PHSRN peptide/peptide gradient surfaces. Peptide PHSRN was found to synergistically enhance cell adhesion at the position where these two ligands are presented in equal amounts, while these peptide ligands were competitively involved in cell adhesion at other positions. This strategy of generating a gradient may be further expandable to the development of functional gradient surfaces of various molecules and materials, such as DNA, proteins, growth factors, and nanoparticles, and could therefore be useful in many fields of research and practical applications.

Facile Method for Development of Ligand-Patterned Substrates Induced by a Chemical Reaction

Patterned substrates have been widely used in various applications, including arrays of biomolecules and cells, highthroughput assays, and direct target sensing. In practice, those demands have been achieved by either of or a combination of two strategies: 1) direct incorporation of biomolecules or functional-group-containing molecules into desired patterns and 2) generation of functional-group-presenting patterns by way of chemical conversions on the surface. The former encompasses microcontact printing (mCP), dip-pen nanolithography (DPN), polymer-pen lithography (PPL), microfluidic networks (mFNs), and microarrays. The latter utilizes the “turning-on” strategy, in which inactive substrates are switched to an active state to reveal organic functional groups, in most cases by electrochemical or photochemical conversions. Patterned functional groups in both strategies are further used as chemical handles for immobilization of biomolecules, such as cell-adhesion ligands, enzyme substrates, proteins, oligosaccharides, and oligonucleotides, to afford patterned substrates. As a typical recent example, Rozkiewicz et al. reported on modified mCP for the preparation of oligonucleotide micropatterns. In their report, oxidized PDMS stamps were first coated with positively charged dendrimers followed by negatively charged oligonucleotides in a layer-by-layer arrangement, and were transferred to a solid support for the generation of microarrays. Smith and co-workers introduced a photo-labile protecting group to a thiol functionality. Various patterns of small molecules and proteins were prepared by using a photolithographic method in combination with thiol-specific conjugation chemistry. Yousaf et al. showed that ligand density and composition influence the rate of stem-cell differentiation by using hydroquinone-based electroactive substrates, which were patterned with a variety of ligands by using microarray technology. Although these two strategies are reliable, well established, and, therefore, widely used, each of the strategies offers limitations on practical use as a general platform for ligand-patterned substrates. For instance, direct contact printing methods, such as mCP, cannot control ligand density on the surface, which can provide important quantitative information for use in experimental design. A concern with regard to the turning-on strategy is that in some cases activated functional groups require specified conjugation chemistry and, therefore, necessitate preparatory steps (tagging steps) to make the ligands compatible with the conjugation reaction. Herein, we describe a simple, efficient, and straightforward method for ligand patterning on a surface, induced by a non-invasive organic chemical reaction—which we have termed a chemical-reaction-induced patterning (CRIP)— and equipped with the capability for control of ligand density. In addition, our method is compatible with common patterning tools and conjugation chemistry. Herein, we demonstrate our strategy by using two popular patterning tools, mCP and microarray, and verify the fidelity of the preparation of ligand-patterned substrates by patterning cell-adhesion ligands and aptamers. Our strategy for preparation of ligand-patterned substrates relies on conversion of a substrate from a dormant (inactive) state to an active state by way of a chemical reaction grafted with a patterning method (Figure 1). The resulting patterned area presents a chemically reactive functional group, that is, a primary amine, which can be harnessed for the immobilization of biomolecules of interest. Our approach utilizes self-assembled monolayers (SAMs) on gold terminated with masked functional groups. Figure 1B shows the structure of the monolayer and the chemical reactions taking place on the surface for the preparation of the amine-functionalized substrate (for the synthesis of the quinone-terminated alkanethiol, see the Supporting information). The quinone-presenting monolayer was treated with a reducing agent by using patterning tools. Upon reduction of the quinone to the corresponding hydroquinone, a cyclization reaction ensues to afford an amine group. The resulting amine can react with linkers or ligands through aminespecific bioconjugation chemistry. The surrounding tri(ethylene glycol) groups provide inertness towards the nonspecific adsorption of proteins and cells, which is the most demanding feature of substrates in biological/biochemical studies at interfaces. Herein, we demonstrate this CRIP strategy by preparing various patterns of RGD (Arg-Gly-Asp) [a] H. Seo, I. Choi, J. Lee, Dr. S. Kim, Prof. D.-E. Kim, Prof. W.-S. Yeo Department of Bioscience and Biotechnology Konkuk University, Seoul, 143-701 (Korea) Fax: (+82) 2-2030-7890 E-mail : wsyeo@konkuk.ac.kr [b] S. K. Kim Nano-Bio Research Center Korea Institute of Science and Technology, Seoul, 136-791 (Korea) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201100084.

Determination of self-exchange rate of alkanethiolates in self-assembled monolayers on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In this paper, we describe a new method for determining the exchange rates of alkanethiolates in self-assembled monolayers (SAMs) on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the compositions of the alkanethiolate in SAMs rapidly and directly. In particular, to investigate the self-exchange of alkanethiols, we prepared a deuterated alkanethiol that has the same molecular properties as the non-deuterated alkanethiol but a different molecular weight. SAMs consisting of deuterated alkanethiolates were immersed in a solution of the non-deuterated alkanethiol, and the influences of the immersion time, temperature, concentration, and solvent on the self-exchange rates were investigated. Furthermore, we assessed the exchange rates among alkanethiols with different carbon chain lengths and different size of ethylene glycol units. In addition, we performed molecular dynamics simulations using a model SAM system in order to understand the molecular mechanism of the exchange process.

Complementary analysis of curcumin biodistribution using optical fluorescence imaging and mass spectrometry

In this study, a complementary analysis was performed to improve the precision of the determination of in vivo curcumin biodistribution after intravenous administration. Overall, similar curcumin biodistribution profiles were obtained using optical fluorescence imaging and mass spectrometry. Poor curcumin accumulation was observed in the heart, spleen, and kidney. However, noticeable accumulation of curcumin in the brain was only observed using fluorescence imaging, probably owing to the insufficient extraction of curcumin from the brain for mass spectrometry. In addition, an exact and reliable measurement of curcumin accumulation in tissues such as the liver, gallbladder, and pancreas was performed using mass spectrometry because of high autofluorescence. Taken together, complementary analysis using optical fluorescence imaging and mass spectrometry allowed the precise determination of curcumin in each tissue. Furthermore, this complementary analytical strategy could be used to elucidate the in vivo distribution of a wide range of fluorescent polyphenols.

Analysis of the biodistribution of natural products in mice by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Natural products originating from plants have various beneficial pharmacological effects, such as anticancer, antimicrobial, and anti-inflammatory activities, while being nontoxic. Therefore, tremendous efforts have been invested in understanding their bioactivities in the body to facilitate therapeutic target validation. However, such research is still challenging for certain natural products, such as flavonoids, which are rapidly metabolized in and eliminated from the human body. To investigate the bioactivities of such products, particularly in certain tissues, it is necessary to understand their biodistribution in vivo. In this respect, reliable analytical methods with simple and efficient procedures for the in vivo evaluation of natural small molecules are urgently required. In particular, mass spectrometry (MS) can be effectively used to analyze small molecules after tissue extraction, as MS has various advantages including accuracy, simplicity, and high sensitivity. Herein, we report the biodistribution of a natural small molecule by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). After intravenously injecting gomisin H into a mouse as a model natural product, it was extracted from each organ and then analyzed by MALDI-TOF MS. The analysis showed that gomisin H accumulated mainly in the liver and relatively large amounts of the product existed in the kidney and brain compared to those in other tissues.

On-Demand Modulation of Bacterial Cell Fates on Multifunctional Dynamic Substrates

This paper reports unprecedented dynamic surfaces based on zwitterionic low-density self-assembled monolayers (LDSAMs) of alkanethiolates on gold, which integrate three interconvertible states-bacteria-adherable, bactericidal, and nonfouling states-through electrical modulations. The conformations of alkanethiolates were electrically modulated to generate zwitterionic, anionic, and cationic surfaces, which responded differently to bacteria and determined the fate of bacteria. Furthermore, the reversible switching of multifunctions of the surface was realized for killing bacteria and subsequently releasing dead bacteria from the surface. For practical application of our strategy, we examined the selective antibacterial effect of our surface for eradication of mycoplasma contaminants in contaminated mammalian cell cultures.

Zinc Ion-immobilized Magnetic Microspheres for Enrichment and Identification of Multi-phosphorylated Peptides by Mass Spectrometry

The selective isolation of phosphorylated peptides and subsequent analysis using mass spectrometry is important for understanding how protein kinase and phosphatase signals can precisely modulate the on/off states of signal transduction pathways. However, the isolation and detection of multi-phosphorylated peptides is still limited due to their distinct affinity to various materials and their poor ionization efficiency. Here, we report a highly efficient and selective enrichment of phosphorylated peptides using binuclear Zn2+-dipicolylamine complex-coated magnetic microspheres (ZnMMs). ZnMMs can utilize the rapid and selective isolation/enrichment of phosphorylated peptides and the subsequent mass spectrometric analysis, given the intrinsic magnetic property of magnetic microspheres and the highly selective binding ability of the binuclear Zn2+-dipicolylamine complex to phosphate groups. α-Casein and β-casein were chosen for a proof-of-concept demonstration. We contemplated that phosphopeptides were selectively isolated and enriched from both the tryptic digests of casein proteins and mixed samples with a high degree of sensitivity by facilitating ZnMMs. Especially, ZnMMs showed high efficiency with multi-phosphopeptides, which are in general difficult to be examined by mass analysis on account of their poor ionization efficiency. For the model protein α, β-casein mixture of the tryptic digest, 17 phosphopeptides were identified with ZnMMs and 82% of the enriched phosphopeptides were multi-phosphorylated peptides, indicating that ZnMMs have excellent enrichment efficiency and strong affinity towards multi-phosphorylated peptides.

On-chip enzymatic assay for chloramphenicol acetyltransferase using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Herein, we report a chloramphenicol (CAP) acetyltransferase (CAT) activity assay based on self-assembled monolayers on gold as an alternative to conventional CAT reporter gene assay systems, which sometimes require toxic materials and complicated steps that limit their use. A CAP derivative presented on a monolayer was converted to the acetylated CAP by CAT in the presence of acetyl-CoA. The conversion was directly monitored by observing the molecular weight changes in CAP using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CAT activity was determined under various reaction conditions by changing reaction times, CAT and acetyl-CoA concentrations. As a practical application, we identified gene expression in bacteria that were transformed with pCAT plasmid DNA. Our strategy can provide a simple and rapid assay that eliminates some commonly used but potentially detrimental steps in enzymatic assays, such as radioactive labeling and complicated separation and purification of analytes prior to detection.