Label-Free Cancer Stem-like Cell Assay Conducted at a Single Cell Level Using Microfluidic Mechanotyping Devices.

The mechanical phenotype of cells is an intrinsic property of individual cells. In fact, this property could serve as a label-free, non-destructive, diagnostic marker of the state of cells owing to its remarkable translational potential. A microfluidic device is a strong candidate for meeting the demand of this translational research as it can be used to diagnose a large population of cells at a single cell level in a high-throughput manner, without the need for off-line pretreatment operations. In this study, we investigated the mechanical phenotype of the human colon adenocarcinoma cell, HT29, which is known to be a heterogeneous cell line with both multipotency and self-renewal abilities. This type of cancer stem-like cell (CSC) is believed to be the unique originators of all tumor cells and may serve as the leading cause of cancer metastasis and drug resistance. By combining consecutive constrictions and microchannels with an ionic current sensing system, we found a high heterogeneity of cell deformability in the population of HT29 cells. Moreover, based on the level of aldehyde dehydrogenase (ALDH) activity and the expression level of CD44s, which are biochemical markers that suggest the multipotency of cells, the high heterogeneity of cell deformability was concluded to be a potential mechanical marker of CSCs. The development of label-free and non-destructive identification and collection techniques for CSCs has remarkable potential not only for cancer diagnosis and prognosis but also for the discovery of a new treatment for cancer.

Tunable Liquid Chromatographic Separation of H/D Isotopologues Enabled by Aromatic π Interactions.

We report hydrogen/deuterium (H/D) isotope effects based on weak intermolecular interactions with polar functional groups and aromatic rings in liquid chromatography (LC). Various LC experiments with different aromatic analytes, separation media, and nonpolar mobile phases were conducted under normal phase LC conditions, where the hydrophobic interaction was completely suppressed. The separation media that had polar functional groups, such as silanol groups, allowed for higher separation efficiencies for the pairs of aromatic H/D isotopologues. In comparing the 13C NMR spectra of protiated and deuterated aromatic analytes, the electron density of the deuterated analyte was found to be slightly higher than that of the protiated analytes. In the case of silanol functional groups, aromatic rings of the analyte acted as donors through the OH-π interaction to hydrogen atoms in the silanol groups. Thus, the deuterated analytes were able to be greatly retained by the stronger OH-π interactions. Furthermore, a C70-fullerene bonded monolithic column (C70 column), which effectively provides CH-π interactions, allowed the opposite isotope effect. Briefly, an electrostatic attraction based on the dipole-(induced) dipole interaction dominated in the CH-π interactions, according to a van't Hoff analysis. Hence, the bonding lengths of the C-H or D bonds were sensitively affected, such that we were able to conclude that the CH-π interaction depended on the geometric effect. Applying these opposing H/D isotope effects, we were able to finally demonstrate effective H/D isotopologue separations by utilizing the complementary action of the OH-π and CH-π interactions.

Differentiating π Interactions by Constructing Concave/Convex Surfaces Using a Bucky Bowl Molecule, Corannulene in Liquid Chromatography.

Convex-concave π conjugated surfaces in hemispherical bucky bowl such as corannulene (Crn) have shown increasing utility in constructing self-assembled new functional materials owing to its unique π electrons and strong dipole. Here, we investigate these specific molecular recognitions on Crn by developing new silica-monolithic capillary columns modified with Crn and evaluating their performance in the separation of different aromatic compounds by liquid chromatography (LC). We synthesized two Crn derivatives and conjugated them onto the surface of a silica monolith. The first Crn derivative was edge functionalized, which can undergo free inversion of a convex-concave surface. The second Crn derivative was synthesized by modifying the spoke of Crn, which suppresses the convex-concave inversion. Results of LC suggest that each surface showed different shape recognition based on π interaction. Furthermore, the concave surface of Crn showed strong CH-π interaction with a planar molecule, coronene, demonstrated by the shifts of the 1H NMR signals of both Crn and coronene resulting from the multiple interactions between Crn and π electrons in coronene. These results clearly demonstrated the presence of CH-π interactions at multiple points, and the role of shape recognition.

Simple and effective label-free capillary electrophoretic analysis of sugars by complexation using quinoline boronic acids.

An effective separation and detection procedure for sugars by capillary electrophoresis (CE) using a complexation between quinolineboronic acid (QBA) and multiple hydroxyl structure of sugar alcohol is reported. We investigated the variation of fluorescence spectra of a variety of QBAs with sorbitol at a wide range of pH conditions and then found that 5-isoQBA strongly enhanced the fluorescence intensity by the complexation at basic pH conditions. The other sugar alcohols having multiple hydroxyls also revealed the enhancement of the fluorescence intensity with 5-isoQBA, whereas the alternation of the intensity was not found in the sugars such as glucose. After optimization of the 5-isoQBA concentration and pH of the buffered solution in CE analysis, 6 sugar alcohols were successfully separated in the order based on the formation constants with 5-isoQBA, which were calculated from the variation of the fluorescence intensity with each sugar alcohol and 5-isoQBA. Furthermore, the limits of detection for sorbitol and xylitol by the CE method were estimated at 15 and 27 µM, respectively.

Unique Separation Behavior of a C60 Fullerene-Bonded Silica Monolith Prepared by an Effective Thermal Coupling Agent.

Herein, we report a newly developed C60 fullerene-bonded silica monolith in a capillary with unique retention behavior due to the structure of C60 fullerene. N-Hydroxysuccinimide (NHS)-conjugated C60 fullerene was successfully synthesized by a thermal coupling agent, perfluorophenyl azide (PFPA), and assigned by spectroscopic analyses. Then, NHS-PFPA-C60 fullerene was attached onto the surface of a silica monolith in a capillary. The capillary provided specific separation ability for polycyclic aromatic hydrocarbons in liquid chromatography by an effective π-π interaction. Furthermore, corannulene, which has a hemispherical structure, was selectively retained in the capillary based on the specific structural recognition due to the spherical C60 fullerene. This is the first report revealing the spherical recognition ability by C60 fullerene in liquid chromatographic separation.

Quantitative ligand immobilization using alginate hydrogel formed in a capillary: application for online affinity concentration.

To simplify a quantitative immobilization procedure of ligands with maintaining their activities, we developed an automated preparation method using an alginate hydrogel partially formed in a capillary. After a sodium alginate solution containing a ligand was injected into the capillary, a background solution containing Ca(2+) was then introduced into the sodium alginate solution zone by applying an appropriate voltage for the hydrogelation, resulting in encapsulation of the ligand by the formed alginate hydrogel. According to the estimated binding capacity for biotin by the encapsulated avidin, the injected avidin was immobilized quantitatively by the formed hydrogel with keeping its affinity. When avidin (3.5-35.2 ng) was immobilized by the proposed method, the immobilization efficiency was estimated to be almost 100%. Furthermore, by using the prepared capillary, biotinylated fluorescein was specifically trapped and separated due to the affinity of the encapsulated avidin. By the change of pH of the background solution, the concentrated analytes could be easily eluted, resulting in 90% recovery with high reproducibility by using 1.18 fmol biotinylated sample.

Antioxidants encapsulated milk-derived exosomes for functional food development.

Reactive oxygen species are known to be involved in various diseases, and antioxidant ingredients are expected to essentially prevent diseases and contribute to improving health. However, antioxidants are easily degraded by enzymes before being absorbed in the intestine, so a means of transport that prevents their degradation in the body is necessary. Exosomes, which play an important role in communication between individual cells, have attracted attention as a new transport carrier of miRNA and DNA, but not yet fully exploited in food research. More recently, exosomes extracted from bovine milk began to be widely used as a cost-effective transport carrier not in clinical medicine but also in functional food materials. To develop practical applications as carriers for functional foods, systematic studies are necessary to clarify the introduction efficiency and the properties of encapsulated substances. In this study, we applied electroporation and incubation to encapsulate antioxidants into the exosomes and studied the encapsulation efficiency into the exosomes and the anticancer activity.

Development of a particle packed bed model for homogeneity evaluation of liquid chromatography column.

Particle packed columns used for liquid chromatography (LC) can suppress a column internal band broadening (hereinafter referred to as band broadening) by packing monodisperse particles homogenously. However, a quantitative evaluation for the effects of particle shape and packed state on band broadening needs to be more investigated. In this study, we fabricated a model of particle packed bed using microfluid LC columns that have pillar array structure prepared by microfabrication technology, evaluating how structural factors inside of a column affect its band broadening. At first, microfluid LC columns was prepared using Si-quartz glass (Si-Q column) for the optimization of LC measurement system. Through the evaluation, it showed 11.6 times higher pressure tolerance compared to that of PDMS-soda lime glass (PDMS-g column). Then, an optimized LC measurement system was constructed using a microfluidic LC column made of Si-Q column, and it was confirmed that the measurement error was small enough and the LC measurement could be performed with high repeatability. Additionally, the effect of a distribution of structural size on band broadening was evaluated. It was confirmed that wide distribution of the structural size provided large band broadening in actual measurements. Comparing two columns having different structural log-normal distributions of 0 and 0.22 showed approximately 1.8 times difference in both real LC measurement. Lastly, the relationship between packed state and band broadening was evaluated. As packed state, we employed void arrangement and structural arrangement in the columns. Different location arrangements of 50 and 100 µm pillar sizes afforded different band broadening. Well-homogenized array showed approximately two times worse band broadening compared to that of delocalized array. Based on these results, the developed packed bed of particles model was able to evaluate the relation between structural factors and band broadening.

Inkjet injection of DNA droplets for microchannel array electrophoresis.

We demonstrated DNA droplets could be injected with an inkjet injector for microchannel array electrophoresis and attained high throughput analysis of biomolecules. This injection method greatly reduced both analysis time and sample amount, compared with a conventional microchip electrophoresis method, and allowed high parallelization of a microchannel array on a small substrate. Since we do not need to use complicated electric programs or microchannel design, our injection method should facilitate omics analyses and contribute to high performance clinical assays.

Development of a microfluidic dispensing device for multivariate data acquisition and application in molecularly imprinting hydrogel preparation.

Molecularly imprinted polymers (MIPs) are superior materials with a molecular recognition ability that apply to various applications. In order to get high specificity recognition for target molecules, selecting polymerization conditions, which provide high interaction with the target template, is crucial. However, it requires time and labor to find the optimal polymerization composition, especially for large biomolecules. The advance in the microfluidic field enables researchers to control the flow rate and divide solutions based on the design of microfluidic devices for acquiring multivariate data by simultaneously preparing samples with different conditions. In this work, we fabricated microfluidic dispensing devices with different flow path widths that can give the solution of different flow rates. The accuracy of the flow rate was compared with the simulation value. As a result, the flow rate data showed almost the same data as the simulation value, and the dispensing volume ratio showed high reproducibility. Besides, the multivariate data from mixing the fluorescent molecule and protein solutions prepared by the dispensing device and a micropipette showed no significant difference with existing laboratory equipment. Finally, the dispensing device was used for preparing MIP hydrogels for lysozyme as a template protein. We successfully acquired multivariate data on the adsorption capacity of proteins, as a result, the hydrogels provided a high imprinting factor and adsorption specificity toward lysozymes.

Simple chemical detection based on a surface-modified electroosmotic pump via interval immobilization.

Environmental water quality monitoring plays an important role in human health risk assessments for pharmaceuticals in water and pollutant source control. A new chemical detection method was developed to enhance molecular selectivity and portability by combining the molecularly imprinted technique and an electroosmotic pump (EOP), which requires only a small pump, batteries and stopwatch in principle. Selective chemical adsorption on the surface-modified EOP decreases the pumping performance of EOP due to a decrease in the surface electric charge. For proof of concept, the microfabricated EOPs with chemical surface treatment were used to investigate the effects of surface chemical change on pumping performance. The microfluidic EOP of a size of 20 mm × 20 mm × 1 mm was modified by an interval immobilization method using the template of 4-(tributylammonium-methyl)-benzyltributylammonium chloride (TBTA) and evaluated by measuring EOF. The pumping performance of the surface-modified EOP was decreased by the selective adsorption of TBTA to a two-point recognition site on the EOP surfaces. The relationships between the flow rate and the TBTA concentration were fitted to the Langmuir equation. The EOP can selectively detect the model substance even in a mixture solution with a different chemical compound. This molecular imprinted EOP does not require large and expensive instruments for driving the device and chemical detection, which can be applied to a portable analytical device for onsite analysis.

Fluorescent detection of target proteins via a molecularly imprinted hydrogel.

Proteins are typically separated by an immune reaction, such as an enzyme-linked immunosorbent assay, and are detected by selective fluorescent labeling. This has potential for complicated procedures and the denaturation of proteins by labeling, and is cost consuming. In this study, we propose a technique for the selective separation and detection of a target protein using a molecularly imprinted hydrogel (PI gel) with fluorescent monomers. We focused on 8-anilino-1-naphthalenesulfonic acid (ANS), where the fluorescence intensity is easily changed by the interaction with proteins, and successfully synthesized the ANS monomer and a poly(ethylene glycol) (PEG) conjugated ANS monomer. The PI gel with the ANS monomers using bovine serum albumin (BSA) as a template showed the selective adsorption of BSA and the fluorescence intensity increased due to the adsorption of BSA.

Separation of saccharides using fullerene-bonded silica monolithic columns via π interactions in liquid chromatography.

We report on a potential method to separate sugars by using the specific interaction between fullerenes and saccharides in liquid chromatography (LC). Aromatic rings with high electron density are believed to interact strongly with saccharides due to CH-π and/or OH-π interactions. In this study, the fullerene-bonded columns were used to separate saccharides by LC under aqueous conditions. As a result, 2-aminobenzamide-labeled glucose homopolymer (Glcs) was effectively separated by both C60 and C70 columns in the range of Glc-1 to Glc-20 and high blood glucose level being retained in greater quantity. Furthermore, similar separations were identified by LC-mass spectrometry with non-labeled glucose homopolymers. Theoretical study based on molecular dynamics and DFT calculation demonstrated that a supramolecular complex of saccharide-fullerene was formed through CH-π and/or OH-π interactions, and that the interactions between saccharide and fullerene increase with the increase units of the saccharide. Additionally, the C60 column retained disaccharides containing maltose, trehalose, and sucrose. In this case, it was assumed that the retention rates were determined by the difference of the dipole moment in each saccharide. These results suggest that the dipole-induced dipole interaction was dominant, and that maltose-with the higher dipole moment-was more strongly retained compared to other disaccharides having lower dipole moment.

Separation of halogenated benzenes enabled by investigation of halogen-π interactions with carbon materials.

The halogen-π (X-π) interaction is an intermolecular interaction between the electron-poor region of bonded halogen atoms and aromatic rings. We report an experimental evaluation of the halogen-π (X-π) interaction using liquid chromatography with carbon-material coated columns providing strong π interactions in the normal phase mode. A C70-fullerene (C70)-coated column showed higher retentions for halogenated benzenes as the number of halogen substitutions increased as a result of X-π interactions. In addition, the strength of the X-π interaction increased in the order of F < Cl < Br < I. Changes to the UV absorption of C70 and the brominated benzenes suggested that the intermolecular interaction changed from the π-π interaction to X-π interaction as the number of bromo substitutions increased. Computer simulations also showed that the difference in dipole moments among structural isomers affected the strength of the π-π interaction. Furthermore, we concluded from small peak shifts in 1H NMR and from computer simulations that the orbital interaction contributes to the X-π interactions. Finally, we succeeded in the one-pot separation of all isomers of brominated benzenes using the C70-coated column by optimizing the mobile phase conditions.

Magnetic Field Stimuli-Sensitive Drug Release Using a Magnetic Thermal Seed Coated with Thermal-Responsive Molecularly Imprinted Polymer.

A new stimulus-responsive drug delivery system using Fe3O4 nanoparticles coated with molecularly imprinted polymer (MIP) is reported. Magnetic thermal seeds (MTS) with their size controlled between 10 and 20 nm that could generate heat under an alternate current (AC) magnetic field were modified with a thermal-responsive MIP by grafting polymerization for effective release of an anticancer drug, methotrexate (MTX). The MIP-coated MTS showed the superparamagnetic property as well as the selective adsorption ability toward MTX, and 80% of MXT adsorbed on the MIP-coated MTS was stimulus released at 60 °C by cleaving hydrogen bonding in the recognition sites. Finally, the MTX release from the MTX-loaded MIP-coated MTS under an AC magnetic field within 10 min was successfully demonstrated.

Efficient extraction of estrogen receptor-active compounds from environmental surface water via a receptor-mimic adsorbent, a hydrophilic PEG-based molecularly imprinted polymer.

We report an efficient screening procedure for the selective detection of compounds that are actively bound to estrogen receptor (ER) from environmental water samples using a receptor-mimic adsorbent prepared by a molecularly imprinted polymer (MIP). To mimic the recognition ability of ER, we improved the typical MIP preparation procedure using a hydrophilic matrix with a polyethylene glycol (PEG)-based crosslinker and a hydrophobic monomer to imitate the hydrophobic pocket of ER. An optimized MIP prepared with methacrylic acid as an additional functional monomer and estriol (E3), an analogue of 17ß-estradiol (E2), exhibited highly selective adsorption for ER-active compounds such as E2 and E3, with significant suppression of non-specific hydrophobic adsorption. The prepared MIP was then applied to the screening of ER-active compounds in sewage samples. The fraction concentrated by the MIP was evaluated by in vitro bioassay using the yeast two-hybrid (Y2H) method and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOFMS). Compared to an authentic adsorbent, styrene-divinylbenzene (SDB)-based resin, the fraction concentrated by the MIP had 120% ER activity in the Y2H assay, and only 25% peak volume was detected in LC-Q-TOFMS. Furthermore, a few ER-active compounds were identified only from the fraction concentrated by the MIP, although they could not be determined in the fraction concentrated by the SDB-based resin due to ion suppression along with high levels of hydrophobic compounds. These results indicated that the newly developed MIP effectively captured ER-active compounds and while allowing most non-ER-active compounds to pass through.

Engineering Nanowire-Mediated Cell Lysis for Microbial Cell Identification.

Researchers have demonstrated great promise for inorganic nanowire use in analyzing cells or intracellular components. Although a stealth effect of nanowires toward cell surfaces allows preservation of the living intact cells when analyzing cells, as a completely opposite approach, the applicability to analyze intracellular components through disrupting cells is also central to understanding cellular information. However, the reported lysis strategy is insufficient for microbial cell lysis due to the cell robustness and wrong approach taken so far ( i. e., nanowire penetration into a cell membrane). Here we propose a nanowire-mediated lysis method for microbial cells by introducing the rupture approach initiated by cell membrane stretching; in other words, the nanowires do not penetrate the membrane, but rather they break the membrane between the nanowires. Entangling cells with the bacteria-compatible and flexible nanowires and membrane stretching of the entangled cells, induced by the shear force, play important roles for the nanowire-mediated lysis to Gram-positive and Gram-negative bacteria and yeast cells. Additionally, the nanowire-mediated lysis is readily compatible with the loop-mediated isothermal amplification (LAMP) method because the lysis is triggered by simply introducing the microbial cells. We show that an integration of the nanowire-mediated lysis with LAMP provides a means for a simple, rapid, one-step identification assay (just introducing a premixed solution into a device), resulting in visual chromatic identification of microbial cells. This approach allows researchers to develop a microfluidic analytical platform not only for microbial cell identification including drug- and heat-resistance cells but also for on-site detection without any contamination.

Quantitative Evaluation of Dielectric Breakdown of Silicon Micro- and Nanofluidic Devices for Electrophoretic Transport of a Single DNA Molecule.

In the present study, we quantitatively evaluated dielectric breakdown in silicon-based micro- and nanofluidic devices under practical electrophoretic conditions by changing the thickness of the insulating layer. At higher buffer concentration, a silicon nanofluidic device with a 100 nm or 250 nm silicon dioxide layer tolerated dielectric breakdown up to ca. 10 V/cm, thereby allowing successful electrophoretic migration of a single DNA molecule through a nanochannel. The observed DNA migration behavior suggested that parameters, such as thickness of the insulating layer, tolerance of dielectric breakdown, and bonding status of silicon and glass substrate, should be optimized to achieve successful electrophoretic transport of a DNA molecule through a nanopore for nanopore-based DNA sequencing applications.

Selective adsorption of carbohydrates and glycoproteins via molecularly imprinted hydrogels: application to visible detection by a boronic acid monomer.

Selective adsorption of carbohydrates and glycoproteins was effectively achieved by molecularly imprinted hydrogels (MIHs) with a poly(ethylene glycol) (PEG)-based crosslinker and 4-vinylphenylboronic acid. In addition, an MIH with a novel boronic acid monomer provided selective adsorption and enabled visible detection of fructose.

Competitive ELISA-like Label-free Detection of Lysozyme by Using a Fluorescent Monomer-doped Molecularly Imprinted Hydrogel.

We report on a molecularly imprinted hydrogel for the selective fluorescent detection of a targeting protein. To achieve the fluorescent detection of proteins, a fluorescent functional monomer was synthesized with fluorescein isothiocyanate and allylamine. The monomer (FITC-AA) provided a gradable alteration of the absorption spectrum, and a decrease of the fluorescent intensity due to an interaction with the model protein, lysozyme. Also, the imprinted hydrogel (PI-gel) was simply prepared with FITC-AA, the anionic functional monomers, poly(ethylene glycol) dimethacrylate, and lysozyme by radical photo polymerization. The PI-gel showed selective adsorption for lysozyme and the imprinting factor reached to 2.7. Additionally, the PI-gel indicated a gradual decrease of the fluorescent intensity corresponding to the lysozyme concertation similar to a competitive enzyme-linked immunosorbent assay detection.