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.

Fluorescence labelled XT5 modified nano-capsules enable highly sensitive myeloma cells detection.

Accurate diagnosis of cancer cells in early stages plays an important role in reliable therapeutic strategies. In this study, we aimed to develop fluorescence-conjugated polymer carrying nanocapsules (NCs) which is highly selective for myeloma cancer cells. To gain specific targeting properties, NCs, XT5 molecules (a benzamide derivative) which shows high affinity properties against protease-activated receptor-1 (PAR1), that overexpressed in myeloma cancer cells, was used. For this purpose, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000]-carboxylic acid (DSPE-PEG2000-COOH) molecules, as a main encapsulation material, was conjugated to XT5 molecules due to esterification reaction using N,N'-dicyclohexylcarbodiimide as a coupling agent. The synthesized DSPE-PEG2000-COO-XT5 was characterized by using FT-IR and1H NMR spectroscopies and results indicated that XT5 molecules were successfully conjugated to DSPE-PEG2000-COOH. Poly(fluorene-alt-benzothiadiazole) (PFBT) conjugated polymer (CP) was encapsulated with DSPE-PEG2000-COO-XT5 due to dissolving in tetrahydrofuran and ultra-sonication in an aqueous solution, respectively. The morphological properties, UV-vis absorbance, and emission properties of obtainedCPencapsulatedDSPE-PEG2000-COO-XT5(CPDP-XT5) NCs was determined by utilizing scanning electron microscopy, UV-vis spectroscopy, and fluorescent spectroscopy, respectively. Cytotoxicity properties of CPDP-XT5 was evaluated by performing MTT assay on RPMI 8226 myeloma cell lines. Cell viability results confirmed that XT5 molecules were successfully conjugated to DSPE-PEG2000-COOH. Specific targeting properties of CPDP-XT5 NCs and XT5-free NCs (CPDP NCs) were investigated on RPMI 8226 myeloma cell lines by utilizing fluorescent microscopy and results indicated that CPDP-XT5 NCs shows significantly high affinity in comparison to CPDP NCs against the cells. Homology modeling and molecular docking properties of XT5 molecules were evaluated and simulation results confirmed our results.

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.

Combining vancomycin-modified gold nanorod arrays and colloidal nanoparticles as a sandwich model for the discrimination of Gram-positive bacteria and their detection via surface-enhanced Raman spectroscopy (SERS).

This study reports the development of a highly sensitive antibiotic-based discrimination and sensor platform for the detection of Gram-positive bacteria through surface-enhanced Raman spectroscopy (SERS). Herein, a combination of gold nanorod arrays (GNAs) and colloidal gold nanoparticles (AuNPs) was used as a SERS platform. To specifically capture Gram-positive bacteria, both GNAs and AuNPs were functionalized with thiol-modified vancomycin (HS-Van) molecules. Three different strains of bacteria (Bacillus subtilis and Staphylococcus aureus as Gram-positive, and Escherichia coli as Gram-negative) were employed to test the proposed system. HS-Van functionalized GNAs (GNA@Van) captured Gram-positive bacteria with high specificity. Also, the bacteria captured by GNA@Van (GNA@Van@Bct) systems showed high signal-to-noise SERS signals with high reproducibility. Addition of AuNP@Van to GNA@Van@Bct resulted in the emergence of a sandwich system (GNA@Van@Bct@Van@AuNP). This system led to a further enhanced SERS signal. The chemometric analysis of GNA@Van@Bct@Van@AuNP enabled the obvious discrimination and detection of Gram-positive bacteria. For comparison, we also tested a smooth gold surface with the same procedure and a similar trend was observed with lower SERS activity.

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.

In vivo imaging/detection of MRSA bacterial infections in mice using fluorescence labelled polymeric nanoparticles carrying vancomycin as the targeting agent.

This study aims to develop fluorescence labelled polymeric nanoparticle (NP) carrying vancomycin as the targeting agent for in vivo imaging of Methicillin-resistant Staphylococcus aureus bacterial infections in animal models. Maleimide functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] as the main was carrier matrix to prepare the NPs. A fluorescence probe, namely, poly[9,9'-bis (6″-N,N,N-trimethylammonium) hexyl) fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide] was encapsulated within these NPs by ultrasonication successfully. UV-Vis spectro- photometry of the NPs showed the characteristic shifting on the peak of conjugated polymers indicating successful packaging of this compound with lipid bilayers in nanoscales. Zeta-sizer and TEM analysis showed that the prepared NPs have a diameter of 80-100 nm in a narrow size distribution. Thiolated vancomycin was synthesized and attached to the NPs as the targeting agent. FTIR and MALDI-TOF spectroscopy analysis confirmed the immobilization. The specific targeting properties of the vancomycin conjugated NPs to the target bacteria were first confirmed in in vitro bacterial cultures in which Escherichia coli was the non-target bacteria - using confocal microscopy and TEM. Imaging of bacterial infections in vivo was investigated in mice model using a non-invasive live animal fluorescence imaging technique. The results confirmed that bacterial infections can be detected using these novel polymeric NPs carrying fluorescence probes for imaging and vancomycin as the targeting agent - in vivo successfully.

Silver or gold deposition onto magnetite nanoparticles by using plant extracts as reducing and stabilizing agents.

In this paper, we describe an environmentally friendly procedure to produce silver (Ag) or gold (Au)-deposited magnetite nanoparticles by using plant extracts (Ligustrum vulgare) as reducing and stabilizing agents. Firstly, magnetite nanoparticles (∼6 nm) with superparamagnetic properties - SPIONs - were synthesized by co-precipitation of Fe(+ 2) and Fe(+ 3) ions. Color changes indicated the differing amounts of Au and Ag ions reduced and deposited on to the SPIONs when the plant extracts were used. UV-vis and transmission electron microscope (TEM) with energy dispersive X-ray (EDX) apparatus confirmed the metallic deposition. Magnetic saturation decreased when the amount of the metallic deposition increased, which was measured by vibrating sample magnetometry (VSM). Due to the molecules coming into contact with - and even remaining on - the surface of the nanoparticles after aggressive washing procedures, the Ag/Au-deposited SPIONs were stable, and almost no agglomeration was observed for months. Fourier Transform Infrared (FTIR) spectra depicted that functional groups such as carboxylic and ketone groups, which are most probably responsible for the reduction and stabilization of Ag/Au- carrying magnetite nanoparticles, originated from the plant extract. The proposed route was facile, viable, and reproducible, and it should be stressed that nanoparticles do contain only safe biomolecules as stabilizing agents on their surfaces.