Fabrication of Perfluoropolyether Microfluidic Devices Using Laser Engraving for Uniform Droplet Production.

A perfluoropolyether (PFPE)-based microfluidic device with cross-junction microchannels was fabricated with the purpose of producing uniform droplets. The microchannels were developed using CO2 laser engraving. PFPE was chosen as the main material because of its excellent solvent resistance. Polyethylene glycol diacrylate (PEGDA) was mixed with PFPE to improve the hydrophilic properties of the inner surface of the microchannels. The microchannels of the polydimethylsiloxane microfluidic device had a blackened and rough surface after laser engraving. By contrast, the inner surface of the microchannels of the PFPE-PEGDA microfluidic device exhibited a smooth surface. The lower power and faster speed of the laser engraving resulted in the development of microchannels with smaller dimensions, less than 30 µm in depth. The PFPE and PFPE-PEGDA microfluidic devices were used to produce uniform water and oil droplets, respectively. We believe that such a PFPE-based microfluidic device with CO2-laser-engraved microchannels can be used as a microfluidic platform for applications in various fields, such as biological and chemical analysis, extraction, and synthesis.

Microfluidic Bioreactor with Fibrous Micromixers for In Vitro mRNA Transcription.

A new type of microfluidic bioreactor with fibrous micromixers for the ingredient mixing and a long macrochannel for the in vitro transcription reaction was fabricated for the continuous production of mRNA. The diameter of the fibrous microchannels in the micromixers was tuned by using an electrospun microfibrous disc with different microfiber diameters. The micromixer with a larger diameter of fibrous microchannels exhibited a better mixing performance than the others. The mixing efficiency was increased to 0.95 while the mixture was passed through the micromixers, suggesting complete mixing. To demonstrate the continuous production of mRNA, the ingredients for in vitro transcription were introduced into the perfluoropolyether microfluidic bioreactor. The mRNA synthesized by the microfluidic bioreactor had the same sequence and in vitro/in vivo performances as those prepared by the bulk reaction. The continuous reaction in the microfluidic bioreactor with efficient mixing performance can be used as a powerful platform for various microfluidic reactions.

Continuous production of lipid nanoparticles by multiple-splitting in microfluidic devices with chaotic microfibrous channels.

Polydimethylsiloxane (PDMS) microfluidic devices with chaotic microfibrous channels were fabricated for the continuous production of lipid nanoparticles (LNPs). Electrospun poly(ε-caprolactone) (PCL) microfibrous matrices with different diameters (3.6 ± 0.3, 6.3 ± 0.4, and 12.2 ± 0.8 µm) were used as a template to develop microfibrous channels. The lipid solution (in ethanol) and water phase were introduced into the microfluidic device as the discontinuous and continuous phases, respectively. The smaller diameter of microfibrous channels and the higher flow rate of the continuous phase resulted in the smaller LNPs with a narrower size distribution. The multiple-splitting of the discontinuous phase and the microscale contact between the two phases in the microfibrous channels were the key features of the LNP production in our approach. The LNPs containing doxorubicin with different average sizes (89.7 ± 35.1 and 190.4 ± 66.4 nm) were prepared using the microfluidic devices for the potential application in tumor therapy. In vitro study revealed higher cellular uptake efficiency and cytotoxicity of the smaller LNPs, especially in the HepG2 cells. The microfluidic devices with microfibrous channels can be widely used as a continuous and high-throughput platform for the production of LNPs containing various active agents.

In Situ Spontaneous Fabrication of Tough and Stretchable Polyurethane-Polyethyleneimine Hydrogels Selectively Triggered by CO2.

CO2 -triggered in situ hydrogels is developed from waterborne poly(ε-caprolactone)-based polyurethane (PU) dispersion and aqueous polyethyleneimine (PEI) solution without any other chemicals and apparatus (e.g., UV light). In the approach, nontoxic CO2 in air is used as a selective trigger for the hydrogel formation. CO2 adsorption onto PEI results in the formation of ammonium cations in PEI and the subsequent multiple ionic crosslinking between PU and PEI chains. Besides the amount of CO2 in air, the rate of hydrogel formation can be controlled by NaHCO3 in the PU-PEI mixture, which serves as a CO2 supplier. The PU hydrogels exhibit tough and stretchable properties with high tensile strength (2.05 MPa) and elongation at break (438.24%), as well as biocompatibility and biodegradability. In addition, the PU hydrogels exhibit high adhesion strength on skin and injectability due to the in situ formation. It is believed that these PU hydrogels have the ideal features for various future applications, such as tissue adhesion barriers, wound dressing, artificial skin, and injectable fillers.

Fabrication of Microfiber-Templated Microfluidic Chips with Microfibrous Channels for High Throughput and Continuous Production of Nanoscale Droplets.

A polydimethylsiloxane (PDMS) microfluidic chip with well-interconnected microfibrous channels was fabricated by using an electrospun poly(ε-caprolactone) (PCL) microfibrous matrix and 3D-printed pattern as templates. The microfiber-templated microfluidic chip (MTMC) was used to produce nanoscale emulsions and spheres through multiple emulsification at many small micro-orifice junctions among microfibrous channels. The emulsion formation mechanisms in the MTMC were the cross-junction dripping or Y-junction splitting at the micro-orifice junctions. We demonstrated the high throughput and continuous production of water-in-oil emulsions and polyethylene glycol-diacrylate (PEG-DA) spheres with controlled size ranges from 2.84 µm to 83.6 nm and 1.03 µm to 45.7 nm, respectively. The average size of the water droplets was tuned by changing the micro-orifice diameter of the MTMC and the flow rate of the continuous phase. The MTMC theoretically produced 58 trillion PEG-DA nanospheres per hour without high shear force. In addition, we demonstrated the higher encapsulation efficiency of the PEG-DA microspheres in the MTMC than that of the microspheres fabricated by ultrasonication. The MTMC can be used as a powerful platform for the large-scale and continuous productions of emulsions and spheres.

Enhanced osteogenic differentiation of alendronate-conjugated nanodiamonds for potential osteoporosis treatment.

BACKGROUND: Alendronate (Alen) is promising material used for bone-targeted drug delivery due to its high bone affinity and therapeutic effects on bone diseases. In addition, Alen can enhance the osteogenic differentiation of osteoblastic cell. Recently, nanodiamonds (NDs) with hardness, non-toxicity, and excellent biocompatibility are employed as promising materials for carrier systems and osteogenic differentiation. Therefore, we prepared Alen-conjugated NDs (Alen-NDs) and evaluated their osteogenic differentiation performances. METHODS: Alen-NDs were synthesized using DMTMM as a coupling reagent. Morphological change of Mouse calvaria-derived preosteoblast (MC3T3-E1) treated with Alen-NDs was observed using the confocal microscope. The osteogenic differentiation was confirmed by cell proliferation, alkaline phosphatase (ALP), calcium deposition, and real-time polymerase chain reaction assay. RESULTS: Alen-NDs were prepared to evaluate their effect on the proliferation and differentiation of osteoblastic MC3T3-E1 cells. The Alen-NDs had a size of about 100 nm, and no cytotoxicity at less than 100 µg/mL of concentration. The treatment of NDs and Alen-NDs reduced the proliferation rate of MC3T3-E1 cells without cell death. Confocal microscopy images confirmed that the treatment of NDs and Alen-NDs changed the cellular morphology from a fibroblastic shape to a cuboidal shape. Flow cytometry, alkaline phosphatase (ALP) activity, calcium deposition, and real-time polymerase chain reaction (RT-PCR) confirmed the higher differentiation of MC3T3-E1 cells treated by Alen-NDs, compared to the groups treated by osteogenic medium and NDs. The higher concentration of Alen-ND treated in MC3T3-E1 resulted in a higher differentiation level. CONCLUSIONS: Alen-NDs can be used as potential therapeutic agents for osteoporosis treatment by inducing osteogenic differentiation.

Organic matrix-free imaging mass spectrometry.

Mass spectrometry (MS) is an ideal tool for analyzing multiple types of (bio)molecular information simultaneously in complex biological systems. In addition, MS provides structural information on targets, and can easily discriminate between true analytes and background. Therefore, imaging mass spectrometry (IMS) enables not only visualization of tissues to give positional information on targets but also allows for molecular analysis of targets by affording the molecular weights. Matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) MS is particularly effective and is generally used for IMS. However, the requirement for an organic matrix raises several limitations that get in the way of accurate and reliable images and hampers imaging of small molecules such as drugs and their metabolites. To overcome these problems, various organic matrix-free LDI IMS systems have been developed, mostly utilizing nanostructured surfaces and inorganic nanoparticles as an alternative to the organic matrix. This minireview highlights and focuses on the progress in organic matrix-free LDI IMS and briefly discusses the use of other IMS techniques such as desorption electrospray ionization, laser ablation electrospray ionization, and secondary ion mass spectrometry. [BMB Reports 2020; 53(7): 349-356].

Polyaniline-grafted nanodiamonds for efficient photothermal tumor therapy.

Polyaniline-grafted nanodiamond (PAN-ND) nanoparticles were fabricated by polymerizing aniline at the surface of amine-modified NDs for efficient photothermal therapy (PTT). A series of PAN from different aniline concentrations were also prepared to compare the properties and the efficiency of PTT. The polymerization rate of aniline was faster in the presence of NDs than that of aniline alone. Compared to PAN nanoparticles, PAN-ND has a spherical shape, smaller size, and ultimately higher cellular uptake efficiency. The temperature of aqueous PAN-ND dispersion increased to 44.4 °C after laser irradiation for 5 min. In addition, the UV absorbance intensity of PAN-ND increased at the lower pH at the near infrared (NIR) region, resulting in an enhanced photothermal effect at a tumor site. Notably, the viability of HeLa cells treated with PAN-ND decreased by less than 20%, suggesting the high efficiency of PTT. The PAN-ND can be a potential candidate for efficient photothermal tumor therapy.

Immobilization of phenol-containing molecules on self-assembled monolayers on gold via surface chemistry.

Various phenol-containing molecules such as flavonoids have a wide range of biological effects including anticancer, antimicrobial, and anti-inflammatory properties, and, therefore, they have become subjects of active research for various medicinal and biological applications. To construct applicable materials incorporated with phenol-containing molecules, strategies for immobilization of phenol-containing molecules on solid substrates are required. Although several immobilization methods have been devised and reported, mostly harnessing phenol functionality, however, development of a general immobilization method has been hampered due to its complicated chemical reactions and low reaction yields on surfaces. Furthermore, the use of phenol as a reaction center may compromise the biological activity of phenol-containing molecules. Here, we describe a simple, fast, and reliable method for the surface immobilization of phenol-containing molecules by introducing chemical functional groups, carboxylic acid, thiol, and azide, while maintaining phenol functionality by way of the Mannich-type condensation reaction. We examined the chemical functionalization of naphthol, tyrosine, and flavanone and their immobilization to the self-assembled monolayers on gold via various surface chemistries: the carbodiimide coupling reaction, Michael addition, and the 'click' reaction. We strongly believe our method can be a general and practical platform for immobilization of various phenol-containing molecules on surfaces of various materials.

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 ß-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, ß-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.

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.

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.

Self-assembled monolayers with dynamicity stemming from (bio)chemical conversions: from construction to application.

Surfaces with "dynamicity" whereby surface properties can be modulated by an external stimulus on user demand have been actively exploited for the past decade. These switchable surfaces with dynamic properties are widely used for a number of applications such as micro/nanoarrays, biomolecule immobilization, basic cell studies, and tissue engineering on a variety of materials. This minireview highlights the dynamic control of surface properties on self-assembled monolayers and focuses on dynamicity that stems from (bio)chemical conversions achieved by electrical potentials, photoillumination, chemical reagents, enzymes, and pH.

The transgenic poplar as an efficient bioreactor system for the production of xylanase.

Plants are attractive expression systems for large-scale, low-cost production of high-value proteins. The xylanase 2 gene (Xyn2), encoding an endo-ß-1,4-xylanase from Trichoderma reesei, was cloned and expressed in Escherichia coli and the poplar (Populus spp.). The optimal temperature and pH of the recombinant xylanase were 50 °C and 5.0 respectively when expressed in E. coli. The purpose of this study was to produce recombinant xylanase in poplar. The Xyn2 gene was transferred into poplars by Agrobacterium-mediated transformation. The transgenic status and transgene expression of the transformed poplar were confirmed by polymerase chain reaction (PCR) genotyping and reverse transcription (RT)-PCR analysis. The poplar-expressed xylanase was biologically active, with an expression level of up to 14.4% of total leaf soluble protein. In the leaves, the average xylanase content was 1.016 mg per g of leaf fresh weight in the transgenic poplar. We found that the poplar might make possible the large-scale production of commercially important recombinant proteins.

On-demand electrochemical activation of the click reaction on self-assembled monolayers on gold presenting masked acetylene groups.

We report on a new surface modification method for grafting a "dynamic" property for on-demand activation of the click reaction. Our approach utilizes the acetylene group masked with dicobalt hexacarbonyl, Co(2)(CO)(6), which is not reactive toward the click reaction. Electrochemical treatment reveals the acetylene group on the selected region, which is then used as a chemical handle for surface functionalization via the click reaction with an azide-containing molecule. Electrochemical and chemical conversions on the surface were verified by cyclic voltammetry, X-ray photoelectron spectroscopy, and fluorescence spectroscopy. We have demonstrated immobilization of an azide-modified RGD peptide and promotion of cell adhesion/migration to the region of electrochemical induction.