Artificial organelles for sustainable chemical energy conversion and production in artificial cells: Artificial mitochondrion and chloroplasts.

Sustainable energy conversion modules are the main challenges for building complex reaction cascades in artificial cells. Recent advances in biotechnology have enabled this sustainable energy supply, especially the adenosine triphosphate (ATP), by mimicking the organelles, which are the core structures for energy conversion in living cells. Three components are mainly shared by the artificial organelles: the membrane compartment separating the inner and outer parts, membrane proteins for proton translocation, and the molecular rotary machine for ATP synthesis. Depending on the initiation factors, they are further categorized into artificial mitochondrion and artificial chloroplasts, which use chemical nutrients for oxidative phosphorylation and light for photosynthesis, respectively. In this review, we summarize the essential components needed for artificial organelles and then review the recent progress on two different artificial organelles. Recent strategies, purified and identified proteins, and working principles are discussed. With more study on the artificial mitochondrion and artificial chloroplasts, they are expected to be very powerful tools, allowing us to achieve complex cascading reactions in artificial cells, like the ones that happen in real cells.

Fabrication of a Tailored, Hybrid Extracellular Matrix Composite.

The extracellular matrix (ECM) is a network of connective fibers that supports cells living in their surroundings. Native ECM, generated by the secretory products of each tissue's resident cells, has a unique architecture with different protein composition depending on the tissue. Therefore, it is very difficult to artificially design in vivo architecture in tissue engineering. In this study, a hybrid ECM scaffold from the basic structure of fibroblast-derived cellular ECMs is fabricated by adding major ECM components of fibronectin (FN) and collagen (COL I) externally. It is confirmed that while maintaining the basic structure of the native ECM, major protein components can be regulated. Then, decellularization is performed to prepare hybrid ECM scaffolds with various protein compositions and it is demonstrated that a liver-mimicking fibronectin (FN)-rich hybrid ECM promoted successful settling of H4IIE rat hepatoma cells. The authors believe that their method holds promise for the fabrication of scaffolds that provide a tailored cellular microenvironment for specific organs and serve as novel pathways for the replacement or regeneration of specific organ tissues.

Pumpless three-dimensional photo paper-based microfluidic analytical device for automatic detection of thioredoxin-1 using enzyme-linked immunosorbent assay.

Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper-based microfluidic analytical device (3D-µPAD; weight: 1.5 g). Unlike conventional paper-based microfluidic analytical devices, the 3D-µPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme's substrate) at the same time in the 3D-µPAD. All the steps can be performed in 14 min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-µPAD, it can detect 0-200 ng/mL of Trx-1, and the prepared 3D-µPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals' blood serum samples using the 3D-µPAD, and comparing results to ELISA, it can be confirmed that the 3D-µPAD is a good tool for cancer diagnosis.

An Accelerated Wound-Healing Surgical Suture Engineered with an Extracellular Matrix.

A suture is a ubiquitous medical device to hold wounded tissues together and support the healing process after surgery. Surgical sutures, having incomplete biocompatibility, often cause unwanted infections or serious secondary trauma to soft or fragile tissue. In this research, UV/ozone (UVO) irradiation or polystyrene sulfonate acid (PSS) dip-coating is used to achieve a fibronectin (FN)-coated absorbable suture system, in which the negatively charged moieties produced on the suture cause fibronectin to change from a soluble plasma form into a fibrous form, mimicking the actions of cellular fibronectin upon binding. The fibrous fibronectin coated on the suture can be exploited as an engineered interface to improve cellular migration and adhesion in the region around the wounded tissue while preventing the binding of infectious bacteria, thereby facilitating wound healing. Furthermore, the FN-coated suture is found to be associated with a lower friction between the suture and the wounded tissue, thus minimizing the occurrence of secondary wounds during surgery. It is believed that this surface modification can be universally applied to most kinds of sutures currently in use, implying that it may be a novel way to develop a highly effective and safer suture system for clinical applications.

Pump-Free Glass-Based Capillary Microfluidic Immuno-Assay Chip for Electrochemical Detection of Prostate-Specific Antigen.

Immuno-assay is one of diagnostic methods that usually measures biomarkers associated with cancers. However, this method is complex and take a long time to analyze. To overcome these disadvantages, many immuno-sensing chips have been designed and developed. However, these devices still require an external pump or electrical source. In this study, our group fabricated a capillary microfluidic device using glass and adhesive polyethylene terephthalate (PET) film, which were designed by simply patterning and cutting to make the microfluidic capillary channels. Using capillary force alone, glass microfluidic chip can control the speed of fluid-flow and the flow sequence by adjusting the width of the channel and design. In addition, each flow can push out other flow without mixing. The glass-based capillary microfluidic chip (GCMC) can automatically perform immunoassay in regular order without external devices and it provide an electrochemical signal analysis in an average of 2 min. The concentration of the prostate-specific antigen (PSA), a biomarker of prostate cancer, was measured by cyclic voltammetry (CV). In conclusion, GCMC can detect between a range of 100 pg/ml to 1 µg/ml of PSA and provide high selectivity to PSA.

Artificial cells containing sustainable energy conversion engines.

Living cells naturally maintain a variety of metabolic reactions via energy conversion mechanisms that are coupled to proton transfer across cell membranes, thereby producing energy-rich compounds. Until now, researchers have been unable to maintain continuous biochemical reactions in artificially engineered cells, mainly due to the lack of mechanisms that generate energy-rich resources, such as adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH). If these metabolic activities in artificial cells are to be sustained, reliable energy transduction strategies must be realized. In this perspective, this article discusses the development of an artificially engineered cell containing a sustainable energy conversion process.

Effects of Silicone Oil on Electrowetting to Actuate a Digital Microfluidic Drop on Paper.

The effects of an immiscible, lubricating polydimethylsiloxane fluid, referred to as silicone oil, on the static deformation and on the dynamic motion of a water drop on paper induced by electrowetting were investigated. The deformation of a drop on a hydrophobic film of amorphous fluoropolymers top-coated with less hydrophobic silicone oil was much more predictable, reversible and reproducible than on the uncoated surface. In the dynamic tribological experiment for a sliding drop along an inclined surface, a significant decrease in the friction coefficient, with an unexpected dependency of the contact area, was observed. Based on the curve fitting analysis, the shear stress and the net friction force were estimated quantitatively. Because of the tribological effect and the reduced shear friction force of the oil film, the static and the dynamic electrowetting states of the water drop were enhanced.

Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system.

Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymerization, with the latter altering outer vesicle morphology. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.

Determining the effects of PEI adsorption on the permeability of 1,2-dipalmitoylphosphatidylcholine/bis(monoacylglycero)phosphate membranes under osmotic stress.

Polycations are used for a number of biological applications, including antibiotics and gene therapy. One aspect of the use of polycation gene carriers such as polyethylenemine (PEI) in gene therapy that is not well understood is their ability to escape from the vesicles they are internalized in. Here, in an attempt to gain a better understanding of PEI interaction with endosomal lipids under osmotic stress, we performed investigations using monolayers and vesicles derived from a mixture of neutral and negative lipids (1,2-dipalmitoylphosphatidylcholine (DPPC) and bis(monoacylglycero)phosphate (BMP), respectively). X-ray reflectivity (XR) and Langmuir trough measurements confirmed PEI adsorption to the negatively charged membrane. Confocal microscopy imaging indicated that PEI adsorption actually increases the overall integrity of the DPPC/BMP vesicle against osmotic stresses while also causing overall deformation and permeabilization of the lipid membrane, thus leading to leakage of contents from the interior of the vesicle. These confocal microscopy observations were also supported by data gathered by dynamic light scattering (DLS). STATEMENT OF SIGNIFICANCE: In recent decades, researchers have investigated polyamine-based gene delivery systems as useful alternatives to viral gene carriers. One step that is crucial to the performance of polyamine gene carriers such as polyethylenemine (PEI) is escape from late endosomal vesicles during intracellular delivery. However, the ability of polyamine/DNA polyplexes to effectively escape from endosomes is a little-understood part of the gene therapy techniques that use these polyplexes. Here, we performed investigations using monolayers and vesicles derived from a mixture of neutral and negative lipids (1,2-dipalmitoylphosphatidylcholine (DPPC) and bis(monoacylglycero)phosphate (BMP), respectively) as model systems for late endosomes in order to examine the interactions of PEI with the DPPC/BMP membranes and study the subsequent effects on the stability and permeability of these membranes.

Micropatterning of mammalian cells on indium tin oxide substrates using ion implantation.

In this study, a simple surface patterning method to create micropatterns of mammalian cells on indium tin oxide (ITO) substrates was developed using ion implantation. Thin polystyrene (PS) films spin-coated on an ITO glass was selectively implanted with accelerated proton ions through a pattern mask and then developed to generate PS micropatterns. Well-organized negative PS patterns were generated on the ITO glass. The results of the in vitro cell culture on the PS-patterned ITO glass with two types of cancer cell lines revealed the formation of well-defined cell patterns through a selective cell adhesion and proliferation only onto the ITO regions separated by PS regions. This facile method for cell patterning may be used to create a desired platform for cellular device applications, such as biosensors and cell microarrays.

Dye-doped silica nanoparticle with HIV-1 TAT peptide for bioimaging.

Multifunctional silica nanoparticles for bioimaging and drug delivery have progressively been developed as novel modalities for cancer diagnosis and treatment. In this study, we synthesized fluorescein isothiocyanate (FITC) doped silica nanoparticle coated with 3-aminopropyl trimethoxysilane (APTS). Additionally, the surface of this silica nanoparticle was modified with HIV-1 trans-activating transcriptional activator (HIV-1 TAT) peptide. The HIV-1 TAT peptide (cell-penetrating peptide) is able to deliver cargos such as oligonucleotides and proteins across the plasma membrane of living cells. This study demonstrates the feasibility of TAT peptide coated silica nanoparticle as a potential carrier for bioimaging and drug delivery.

Patterned immobilization of biomolecules on a polymer surface functionalized by radiation grafting.

Patterned graft polymerization of a functional monomer on a hydrophobic polymer surface was proposed for biomolecule patterning. A poly(vinylidene fluoride) (PVDF) film surface was selectively activated by ion implantation through a pattern mask and acrylic acid (AA) was then graft polymerized onto the activated regions of the PVDF surfaces. The peroxide concentration on the implanted surface depended on the fluence, which had a considerable effect on the grafting degree of AA. Afterwards, amine-functionalized biotin and probe DNA were immobilized on the poly(acrylic acid)-grafted regions of the PVDF surfaces. Specific binding of biotin with streptavidin and hybridization of probe DNA with complimentary DNA proved successful protein and DNA patterning and well-defined 50 microm dot-type patterns of the streptavidin and DNA were obtained. These results confirmed the potential of this strategy for patterning of various biomolecules.

Functionalization of carbon nanotubes by radiation-induced graft polymerization.

Multi-walled carbon nanotubes (MWCNTs) were functionalized by radiation-induced graft polymerization of acrylic acid onto the surface of MWCNTs in order to improve their dispersibility in water. 1H NMR, Raman spectroscopy, TEM, and TGA techniques were used to characterize the resulting functionalized MWCNTs. The grafting degree was dependent on the grafting conditions such as the absorbed dose and the monomer concentration. The experimental results confirmed that poly(acrylic acid) chains were successfully grafted onto the surface of the MWCNTs. The poly(acrylic acid)-grafted MWCNTs showed a much better water dispersibility than the pristine MWCNTs.

X-ray reflectivity study of a transcription-activating factor-derived peptide penetration into the model phospholipid monolayers.

The penetration of a transcription-activating factor (TAT)-derived, cell-penetration peptide onto 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine] (DPPS) monolayer on phosphate-buffered saline subphase was characterized. The surface area at the target pressure increased noticeably by the peptide penetration from the subphase to the phospholipid monolayer, which might suggest a direct penetration of the peptide across the pure phospholipid bilayer membrane. Interestingly, the more significant area increase at 35 mN/m was monitored from DPPC monolayer, contrary to the simple charge interaction: the net neutral DPPC, the net-negative DPPS, and the positive TAT-derived peptides (TDP). X-ray reflectivity measurements as well as the molecular area from pi (surface pressure)-A (area) isotherms suggest that the packing density of DPPS at the target pressure is too high to allow the effective penetration of the peptide into the monolayer and the positively charged peptides can be entrapped at the negative electrostatic well of DPPS headgroup layer, leading to the simple adsorption on the DPPS monolayer instead of penetration into it. Thus, more penetration with less adsorption of the peptide is induced by DPPC monolayer than DPPS monolayer.

Radiation hardness of the electrical properties of carbon nanotube network field effect transistors under high-energy proton irradiation.

The effect of high-energy proton irradiation on the physical properties of carbon nanotubes (CNTs) was investigated. The focus of the study was on the electrical properties of single-walled carbon nanotube (SWNT) network devices exposed to proton beams. Field-effect transistors (FETs) of network type were fabricated using SWNTs and were then irradiated by high-energy proton beams of 10-35 MeV with a fluence of 4 × 10(10)-4 × 10(12) cm(-2) that are comparable to the aerospace radiation environment. The electrical properties of both metallic and semiconducting CNT network FET devices underwent no significant change after the high-energy proton irradiation, indicating that the CNT network devices are very tolerant in proton beams. Raman spectra confirm the proton-radiation hardness of CNT network FET devices. The radiation hardness of CNT network FET devices promises therefore the potential usefulness of CNT-based electronics for future space application.

In situ photopolymerization of a polymerizable poly(ethylene glycol)-covered phospholipid monolayer on a methacryloyl-terminated substrate.

We have prepared a chemically anchored monolayer of PEG (poly(ethylene glycol)) and phospholipid mixture (PEG/phospholipid) on a methacryloyl-terminated substrate by in situ photopolymerization. Both monoacryloyl phospholipid (acryloyl-PC, 1-palmitoyl-2-[12-(acryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine) and monoacryloyl PEG (acryloyl-PEG, 12-(acryloyloxy)dodecanoyl-PEG) were synthesized by modifyingphospholipid and PEGwith 12-(acryloyloxy)-1-dodecanoic acid and 12-(acryloyloxy)-1-dodecanol, respectively. The surface pressure-area (pi-A) isotherm showed that acryloyl-PEG molecules were stable in the phospholipid monolayer and that they could be evenly inserted into a phospholipid monolayer at the air/water interface. By adding 10 mol % acryloyl-PEG into phosholipid vesicles, we could produce a PEG/phosholipid monolayer on methacryloyl-terminated substrates using vesicle fusion for 3 h. Then, this polymerizable PEG/phospholipid monolayer was in situ photopolymerized onto a methacryloyl-terminated substrate with eosin Y/triethanolamine as co-initiators. Optimal vesicle fusion and irradiation condition were determined with respect to the vesicle fusion time and duration of irradiation. As confirmed by atomic force microscopy and X-ray reflectivity studies, the polymerized PEG/phosholipid surface formed a PEG-covered phospholipid monolayer with thicknesses of 3 and 6 nm for the base phospholipid monolayer and the covering PEG layer, respectively. The chemical anchoring efficiency ofpolymerized PEG and phospholipid molecules, which was calculated by the relative carbon ratio of each surface before and after methanol washing using X-ray photoelectron spectroscopy, was 98%. This polymerized PEG/phosholipid monolayer showed good stability in organic solution due to firm chemical anchoring to a solid surface.