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.

Developing a Novel Platelet-Rich Plasma-Laden Bioadhesive Hydrogel Contact Lens for the Treatment of Ocular Surface Chemical Injuries.

Permanent injury to corneal limbal stem cells after ocular surface chemical and thermal injuries is a major cause of corneal blindness. In this study, a PRP-laden GelMA hydrogel contact lens is manufactured which is aimed to support the limbal niche after ocular surface insults thereby preventing limbal stem cell failure. GelMA with varying platelet-rich plasma (PRP) concentrations (5%, 10%, and 20%) is photopolymerized using a visible light crosslinking system followed by characterizations of mechanical properties, growth factor release, enzymatic degradation, and in vitro cytotoxicity. The addition of 10% PRP into 10% GelMA hydrogel precursor solution results in the highest tensile and compressive modulus (38 and 110 kPa, respectively) and burst pressure (251±37.66 mmHg). Degradation time varies according to the concentration of the collagenase enzyme tested (0, 2.5, 5, and 40 µg/mL) and is most prolonged with 20% PRP. EGF and TGF-ß release profiles suggest an initial burst release followed by sustained release, most consistent in the 10% PRP sample. Although cell viability decreases on day 1, rapid recovery is observed and is approximately 120% after day 21. PRP-laden GelMA in the form of a contact lens may be a promising biomaterial-based treatment approach for the maintenance of limbal epithelial stem cells after ocular surface insults.

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.

On-chip label-free impedance-based detection of antibiotic permeation.

Biosensors are analytical tools used for the analysis of biomaterial samples and provide an understanding about the biocomposition, structure, and function of biomolecules and/or biomechanisms by converting the biological response into an electrical and/or optical signal. In particular, with the rise in antibiotic resistance amongst pathogenic bacteria, the study of antibiotic activity and transport across cell membranes in the field of biosensors has been gaining widespread importance. Herein, for the rapid and label-free detection of antibiotic permeation across a membrane, a microelectrode integrated microfluidic device is presented. The integrated chip consists of polydimethylsiloxane based microfluidic channels bonded onto microelectrodes on-glass and enables us to recognize the antibiotic permeation across a membrane into the model membranes based on electrical impedance measurement, while also allowing optical monitoring. Impedance testing is label free and therefore allows the detection of both fluorescent and non-fluorescent antibiotics. As a model membrane, Giant Unilamellar Vesicles (GUVs) are used and impedance measurements were performed by a precision inductance, capacitance, and resistance metre. The measured signal recorded from the device was used to determine the change in concentration inside and outside of the GUVs. We have found that permeation of antibiotic molecules can be easily monitored over time using the proposed integrated device. The results also show a clear difference between bilayer permeation that occurs through the lipidic bilayer and porin-mediated permeation through the porin channels inserted in the lipid bilayer.

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.

Investigation of the Effect of Channel Structure and Flow Rate on On-Chip Bacterial Lysis.

Successful lysis of cells/microorganisms is a key step in the sample preparation in fields like molecular biology, bioengineering, and biomedical engineering. This study therefore aims to investigate the lysis of bacteria on-chip and its dependence on both microfluidic channel structure and flow rate. Effects of temperature on lysis on-chip were also investigated. To perform these investigations, three different microfluidic chips were designed and produced (straight, zigzag and circular configurations), while the length of the channels were kept constant. As an exemplary case, Mycobacterium smegmatis was chosen to represent the acid-fast bacteria. Bacterial suspensions of 1.5 McFarland were injected into the chips at various flow rates (0.6- [Formula: see text]/min) either at room temperature or 50° C. In order to understand the on-chip lysis performance fully, off-chip experiments were carried out at durations which are equal to those bacteria spent in the channel from inlet to the outlet at different flow rates. We also performed COMSOL multiphysics program simulations to evaluate further the effect of the applied parameters. As a result, we found that the structure and the flow rate do not affect lysis over all in all investigated channel types, however on-chip experiments at room temperature produced more effective lysis compared to the on-chip and the off-chip samples performed at higher temperatures. Interestingly on-chip experiments at higher tempratures do not result in effective lysis.

Label-free molecular detection of antibiotic susceptibility for Mycobacterium smegmatis using a low cost electrode format.

Today, the emergence of antibiotic resistance in pathogenic bacteria is considered an important problem for society. Excessive consumption of antibiotics, long-term treatments, and inappropriate prescriptions continually increase the severity of the problem. Improving antibiotic stewardship requires improved diagnostic testing, and, therefore, in vitro antibiotic susceptibility testing is becoming increasingly important. This research details the development of an antibiotic susceptibility test for Mycobacterium smegmatis using streptomycin as antibiotics. This strain was selected because it is a member of the slow growing Mycobacterium genus and serves as a useful surrogate organism for M. tuberculosis. A commercially available and low-cost screen-printed gold electrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome was employed to monitor M. smegmatis nucleic acid sequences using the techniques of square-wave voltammetry and electrochemical impedance spectroscopy. The results show that it was possible to detect M. smegmatis sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. As a result, the in vitro antibiotic susceptibility test revealed that M. smegmatis showed sensitivity to streptomycin after a 24-H incubation, with the developed protocol representing a potential approach to determining antibiotic susceptibility more quickly and economically than current methods.