High-throughput microfluidic chip with silica gel-C18 channels for cyclotide separation.

Over the past two decades, microfluidic-based separations have been used for the purification, isolation, and separation of biomolecules to overcome difficulties encountered by conventional chromatography-based methods including high cost, long processing times, sample volumes, and low separation efficiency. Cyclotides, or cyclic peptides used by some plant families as defense agents, have attracted the interest of scientists because of their biological activities varying from antimicrobial to anticancer properties. The separation process has a critical impact in terms of obtaining pure cyclotides for drug development strategies. Here, for the first time, a mimic of the high-performance liquid chromatography (HPLC) on microfluidic chip strategy was used to separate the cyclotides. In this regard, silica gel-C18 was synthesized and characterized by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) and then filled inside the microchannel to prepare an HPLC C18 column-like structure inside the microchannel. Cyclotide extract was obtained from Viola ignobilis by a low voltage electric field extraction method and characterized by HPLC and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). The extract that contained vigno 1, 2, 3, 4, 5, and varv A cyclotides was added to the microchannel where distilled water was used as a mobile phase with 1 µL/min flow rate and then samples were collected in 2-min intervals until 10 min. Results show that cyclotides can be successfully separated from each other and collected from the microchannel at different periods of time. These findings demonstrate that the use of microfluidic channels has a high impact on the separation of cyclotides as a rapid, cost-effective, and simple method and the device can find widespread applications in drug discovery research.

On chip microfluidic separation of cyclotides.

Cyclotides as a cyclic peptide produced by different groups of plants have been a very attractive field of research due to their exceptional properties in biological activities and drug design applications. The importance of cyclotides as new biological activities from nature caused to attract researchers to develop new separation systems. Recent growth and development on chip-based technology for separation and bioassay especially for anticancer having sparklingly advantages comparison with common traditional methods. In this study, the microfluidic separation of Vigno 1-5 cyclotides extracted from Viola ignobilis by using polar and nonpolar forces as a liquid-liquid interaction was investigated through modified microfluidic chips and then the results were compared with a traditional counterpart technique of high-performance liquid chromatography (HPLC). The traditional process of separating cyclotides from plants is a costly and time-consuming procedure. The scientific novelty of this study is to accelerate the separation of cyclotides using modified microfluidic chips with low cost and high efficiency. The results revealed that a novel and simple microfluidic chip concept is an effective approach for separating the Vigno groups in the violet extract. We believe that the concept could potentially be utilized for further drug development process especially for anticancer studies by coupling bioassay chips as online procedures via reducing in time and cost compared with traditional offline methods.

Effect of microfluidic channel integration onto gold microelectrode on its redox electrochemistry.

Microfluidic systems have attracted significant interest in recent years as they are extensively employed in lab-on-chip and organ-on-chip research. Their combination with electrochemical platforms offers many advantages, promising a high potential for sensing applications, still the microfluidic-channel integration onto electrodes might induce challenges related to changes in signal-to-noise ratios and mass transport conditions. In this study, we investigated the effect of microfluidic channel integration in redox behavior of thermally deposited gold thin film microelectrodes by voltammetric (CV and SWV) electrochemical measurements. Using different dimensions of PDMS microfluidic channels (i.e. widths of 50, 100, 250, and 500 µm) and a constant electrode dimension (200 µm), we analyzed the relationship between altered electroactive area and electrochemical response against target redox molecules. The increases in electroactive area which were determined by the microfluidic channel sizes were in well-correlation with the obtained CV and SWV redox currents as expected. There was no significant decrease in signal-to-noise ratio in microchannel-integrated electrodes. AFM and SEM characterization demonstrated that thermally deposited thin film electrodes had significantly lower (approximately 25 fold) surface roughness in comparison to commercial screen-printed electrodes. Additionally, we have observed a clear microelectrode-to-macroelectrode transition, from hemispherical to linear (planar) diffusion in other terms, with the increasing channel size.

Revealing the single-channel characteristics of OprD (OccAB1) porins from hospital strains of Acinetobacter baumannii.

Nowadays, reports of antimicrobial resistance (AMR) against many antibiotics are increasing because of their misapplication. With this rise, there is a serious decrease in the discovery and development of new types of antibiotics amid an increase in multi-drug resistance. Unfermented Acinetobacter baumannii from gram-negative bacteria, which is one of the main causes of nosocomial infections and multi-drug resistance, has 4 main kinds of antibiotic resistance mechanism: inactivating antibiotics by enzymes, reduced numbers of porins and changing of their target or cellular functions due to mutations, and efflux pumps. In this study, characterization of the possible mutations in OprD (OccAB1) porins from hospital strains of A. baumannii were investigated using single channel electrophysiology and compared with the standard OprD isolated from wild type ATCC 19,606. For this aim, 5 A. baumannii bacteria samples were obtained from patients infected with A. baumannii, after which OprD porins were isolated from these A. baumannii strains. OprD porins were then inserted in an artificial lipid bilayer and the current-voltage curves were obtained using electrical recordings through a pair of Ag/AgCl electrodes. We observed that each porin has a characteristic conductance and single channel recording, which then leads to differences in channel diameter. Finally, the single channel data have been compared with the gene sequences of each porin. It was interesting to find out that each porin isolated has a unique porin diameter and decreased anion selectivity compared to the wild type.

Simple and low-cost antibiotic susceptibility testing for Mycobacterium tuberculosis using screen-printed electrodes.

One quarter of the global population is thought to be latently infected by Mycobacterium tuberculosis (TB) with it estimated that 1 in 10 of those people will go on to develop active disease. Due to the fact that M. tuberculosis (TB) is a disease most often associated with low- and middle-income countries, it is critical that low-cost and easy-to-use technological solutions are developed, which can have a direct impact on diagnosis and prescribing practice for TB. One area where intervention could be particularly useful is antibiotic susceptibility testing (AST). This work presents a low-cost, simple-to-use AST sensor that can detect drug susceptibility on the basis of changing RNA abundance for the typically slow-growing M. tuberculosis (TB) pathogen in 96 h using screen-printed electrodes and standard molecular biology laboratory reactionware. In order to find out the sensitivity of applied sensor platform, a different concentration (108 -103  CFU/mL) of M. tuberculosis was performed, and limit of detection and limit of quantitation were calculated as 103.82 and 1011.59  CFU/mL, respectively. The results display that it was possible to detect TB sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. These findings pave the way for the development of a comprehensive, low-cost, and simple-to-use AST system for prescribing in TB and multidrug-resistant tuberculosis.

Cheap portable electroformed giant unilamellar vesicles preparation kit.

The membrane of a cell separates the internal and external media of the cell and contributes to a variety of important processes, including gradient maintenance and signal transduction. Synthetic lipid-made vesicles are commonly utilized as cell membrane model systems. These could be liposomes or giant unilamellar vesicles (GUVs) in most cases. Liposomes are typically less than 0.5 microns in size, limiting their use for most microscopy experiments. GUVs are a form of liposomes that ranges in size from 5 to 200 microns and are ideal for examining complex phase behaviors of biomembranes using the classical optical setting. This study details the step-by-step development of a portable, light and low-cost kit for generating GUVs by electroformation. Our kit contains an in-built electronic circuitry, and the GUV generation setup, consisting of 3 ITO-coated glasses with heating electrode connections. Approximately 600 µl of GUVs can be produced in one experiment, while the amount could be increased by changing the dimensions of the GUV generation setup. Finally, the originality of the study comes from the fact that many users from different fields unfamiliar with electronics can use our home-built cost-effective approach instead of their expensive commercial counterparts.

Electrochemical-based ''antibiotsensor'' for the whole-cell detection of the vancomycin-susceptible bacteria.

In this study, we aim to develop an antibiotic-based biosensor platform 'Antibiotsensor' for the specific detection of gram-positive bacteria using vancomycin modified Screen Printed Gold Electrodes (SPGEs). Through this pathway, vancomycin molecules were first functionalized with thiol groups and characterized with quadrupole time of flight (q-TOF) mass spectroscopy analysis. Immobilization of thiolated vancomycin molecules (HS-Van) onto SPGEs was carried out based on self-assembled monolayer (SAM) phenomenon. Electrochemical impedance spectroscopy (EIS) was employed to test the detection and showed a considerable change in impedance value upon the binding of HS-Van molecules onto the electrode surface. Atomic Force Microscopy analysis indicated that SPGE was successfully modified upon the treatment with HS-Van molecules based on the shift in surface roughness from 173 ± 2 nm to 301 ± 3 nm. Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy proved the EIS and AFM results as well by showing characteristic peaks of immobilized HS-Van molecule. As a proof-of-concept, EIS-based susceptibility testing was performed using Escherichia coli, Staphylococcus aureus and Mycobacterium smegmatis bacteria to prove the specificity of obtained SPGE-Van. EIS data showed that the charge transfer resistance (Rct) values changed from 1.08, 1.18 to 26.5, respectively, indicating that vancomycin susceptible S. aureus was successfully attached onto SPGE-Van surface strongly, while vancomycin resistance E. coli and M. smegmatis did not show any significant attachment properties. In addition, different concentration (108-10 CFU/mL) of S. aureus was performed to investigate sensitivity of proposed sensor platform. Limit of detection and limit of quantitation was calculated as 101.58 and 104.81 CFU/mL, respectively. Scanning electron microscopy (SEM) analysis also confirmed that only S. aureus bacteria was attached to the surface in a dense monolayer distribution. We believe that the proposed approach is selective and sensitive towards the whole-cell detection of vancomycin-susceptible bacteria and can be modified for different purposes in the future.

Current Strategies for the Regeneration of Skeletal Muscle Tissue.

Traumatic injuries, tumor resections, and degenerative diseases can damage skeletal muscle and lead to functional impairment and severe disability. Skeletal muscle regeneration is a complex process that depends on various cell types, signaling molecules, architectural cues, and physicochemical properties to be successful. To promote muscle repair and regeneration, various strategies for skeletal muscle tissue engineering have been developed in the last decades. However, there is still a high demand for the development of new methods and materials that promote skeletal muscle repair and functional regeneration to bring approaches closer to therapies in the clinic that structurally and functionally repair muscle. The combination of stem cells, biomaterials, and biomolecules is used to induce skeletal muscle regeneration. In this review, we provide an overview of different cell types used to treat skeletal muscle injury, highlight current strategies in biomaterial-based approaches, the importance of topography for the successful creation of functional striated muscle fibers, and discuss novel methods for muscle regeneration and challenges for their future clinical implementation.

Rapid fabrication of teflon apertures by controlled high voltage pulses for formation of free standing planar lipid bilayer membrane.

Free standing artificial lipid bilayers are widely used in the study of biological pores. In these types of studies, the free standing planar lipid bilayer is formed over a micron-sized aperture consisting of either polymer such as Polytetrafluoroethylene (PTFE, Teflon) or glass. Teflon is chemically inert, has a low dielectric constant, and has a high electrical resistance which combined allow for obtaining low noise recordings. This study investigates the reproducible generation of micropores in the range of 50-100 microns in diameter in a Teflon film using a high energy discharge set-up. The discharger set-up consists of a microprocessor, a transformer, a voltage regulator, and is controlled by a computer. We compared two approaches for pore creation: single and multi-pulse methods. The results showed that the multi-pulse method produced narrower aperture size distributions and is more convenient for lipid bilayer formation, and thus would have a higher success rate than the single-pulse method. The bilayer stability experiments showed that the lipid bilayer lasts for more than 33 h. Finally, as a proof-of-concept, we show that the single and multi-channel electrophysiology experiments were successfully performed with the apertures created by using the mentioned discharger. In conclusion, the described discharger provides reproducible Teflon-pores in a cheap and easy-to-operate manner.

Towards understanding single-channel characteristics of OccK8 purified from Pseudomonas aeruginosa.

Antibiotic resistance in Gram-negative bacteria causes serious health issues worldwide. Bacteria employ several resistance mechanisms to cope with antimicrobials. One of their strategies is to reduce the permeability of antibiotics either through general diffusion porins or substrate-specific channels. In this study, one of the substrate-specific channels from Pseudomonas aeruginosa, OccK8 (also known as OprE), was investigated using single-channel electrophysiology. The study also includes the investigation of permeability properties of several amino acids with different charged groups (i.e. arginine, glycine and glutamic acid) through OccK8. We observed four different conformations of the same OccK8 channel when inserted in lipid bilayers. This is in contrast to previous studies where heterologous expressed OccK8 in E. coli showed only one conformation. We hypothesized that the difference in our study was due to the expression and purification of the native channel from P. aeruginosa. The single-channel uptake characteristics of the porin showed that negatively charged glutamic acid preferentially interacted with the channel while the positively charged arginine molecule showed infrequent interaction with OccK8. The neutral amino acid glycine did not show any interaction at the physiological conditions.

Decellularized inner body membranes for tissue engineering: A review.

Body membranes are thin sheets/layers of cells or tissues which cover the surface of internal organs, the outside of the body and lines various body cavities. These membranes are separated into two main groups which are epithelial membranes and connective tissue membranes. Decellularized forms of inner body membranes in the groups of epithelial membranes (amniotic membrane, mesentery, omentum, pericardium, peritoneum, pleura) and connective tissue membranes (fascia, periosteum, synovial membrane) have been used in tissue engineering studies for preparation and regeneration of various tissues such as bone, tendon, cartilage, skin, cornea, ocular surface, uterine, periodontium, vascular and cardiovascular structures. Decellularized inner body membranes have high biocompatibility and support cell attachment, cell growth and angiogenesis which are desired properties for using as versatile tools in tissue engineering applications. Even though, decellularized forms of these membranes have been used in many studies, it is necessary to develop new decellularization methods for more effective cell removal and less destructive properties on tissue structures. Moreover, development of decellularization agents which target removal of antigens of donor tissues is also essential because these antigens are one of the main reasons for tissue-organ rejections in allogeneic and xenogeneic tissue-organ implantations. This review provides comprehensive information and analysis about the current state of the art in the literature on decellularized inner body membranes and applications of these membranes in tissue engineering.

Development of an On-Chip Antibiotic Permeability Assay With Single Molecule Detection Capability.

Electrophysiology is the method of choice to characterize membrane channels. In this paper, we demonstrate a patch pipette based simple miniaturization that allows performing conductance measurements on a planar lipid bilayer in a microfluidic channel. Membrane proteins were reconstituted into Giant Unilamellar Vesicles (GUVs) by electroswelling, and GUVs with a single channel insertion were patched at the tip of pipette. We applied this approach to investigate the interactions of porins from E.coli with single antibiotics, and this will potentially provide information on the permeability rates. The results of this paper suggest that this approach can be extended to the integration of several pipettes into the microfluidic channel from different positions, allowing the multiplexed recordings and also reducing the substrate consumption below volumes.