On-chip cell lysis by antibacterial non-leaching reusable quaternary ammonium monolithic column

Reusable antibacterial non-leaching monolithic columns polymerized in microfluidic channels designed for on-chip cell lysis applications were obtained by the photoinitiated free radical copolymerization of diallyldimethylammonium chloride (DADMAC) and ethylene glycol diacrylate (EGDA) in the presence of a porogenic solvent. The microfluidic channels were fabricated in cross-linked poly(methyl methacrylate) (X-PMMA) substrates by laser micromachining. The monolithic columns have the ability to inhibit the growth of, kill and efficiently lyse gram-positive Micrococcus luteus (Schroeter) (ATCC 4698) and Kocuria rosea (ATCC 186), and gram-negative bacteria Pseudomonas putida (ATCC 12633) and Escherichia coli (ATCC 35218) by mechanically shearing the bacterial membrane when forcing the cells to pass through the narrow pores of the monolithic column, and simultaneously disintegrating the cell membrane by physical contact with the antibacterial surface of the column. Cell lysis was confirmed by off-chip PCR without the need for further purification. The influence of the cross-linking monomer on bacterial growth inhibition, leaching, lysis efficiency of the monolithic column and its mechanical stability within the microfluidic channel were investigated and analyzed for three different cross-linking monomers: ethylene glycol dimethacrylate (EGDA), ethylene glycol dimethacrylate (EGDMA) and 1,6-hexanediol dimethacrylate (1,6-HDDMA). Furthermore, the bonding efficiency of two X-PMMA substrates with different cross-linking levels was studied. The monolithic columns were shown to be stable, non-leaching, and reusable for over 30 lysis cycles without significant performance degradation or DNA carryover when they were back-flushed between lysis cycles

Antibacterial Porous Polymeric Monolith Columns with Amphiphilic and Polycationic Character on Cross-linked PMMA Substrates for Cell Lysis Applications

The application of porous polymeric monolith (PPM) columns as an effective tool for bacterial cell lysi within microfluidic chips is demonstrated. By taking advantage of the large surface area and controllable pore size inherent to PPMs, we developed a double mechanism cell lysis technique. The bacterial cell wall is mechanically sheared by flowing through the porous medium of the PPM column, but it is also damaged and disintegrated by physical contact with the antibacterial polymeric biocide covering the porous surface. This leads to leakage of the intracellular contents. The stable and nonleaching antibacterial column introduced in this work alleviates the need for chemical or enzymatic lysins and their potential release of polymerase chain reaction (PCR) inhibitors. The PPM columns were obtained by the photoinitiated free radical co polymerization of n-butyl methacrylate (BuMA) and N-(tert butyloxycarbonyl)aminoethyl methacrylate (Boc-AEMA) in the presence of a cross-linker and porogenic solvents. The porous network was synthesized directly inside a microfluidic channel fabricated in a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by laser micromachining. After removing the Boc protecting group with phosphoric acid, an amphiphilic and cationic network structure reminiscent of synthetic mimics of antimicrobial peptides (SMAMPs) was obtained. The antibacterial activity of the PPM columns was tested against Bacillus subtilis (B. subtilis) and Escherichia coli (E. coli) cells. Cell lysis was evidenced by DNA release, which was then amplified by PCR and confirmed by gel electrophoresis, to verify that the antibacterial monolithic columns did not strongly interfere with the PCR process

Antibacterial Porous Polymeric Monolith for On-Chip Cell Lysis

The application of porous polymeric monolith (PPM) column as an e effective tool for bacterial cell lysis is demonstrated in this thesis. By exploiting the expansive surface area and controllable pore size inalienable to PPM, a double mechanism cell lysis technique was developed. The bacterial cell wall is mechanically sheared (mechanical-shear lysis) by flowing through the narrow porous medium of the PPM column, but it is also damaged and disintegrated by physical contact (contact-killing lysis) with the antibacterial polymeric biocide covering the porous surface. This leads to leakage of the intracellular contents. The antibacterial monolithic columns possess the aptitude to constrain the growth, kill and e efficiently lyse gram-negative and gram-positive bacterial cells. The developed antibacterial PPM columns can be potentially used in developing commercial macro-columns for cell lysis like the columns commercially available for DNA isolation. Also, they can be used within microfluidic channels for on-chip cell lysis at the heart of integrated sample preparation device. The developed cell lysis technique eff efficiently lysed numerous gram-negative and gram-positive bacterial species with no chemical or enzymatic reagents utilized, power consumption required, complicated design and fabrication processes. Also, the cell lysate containing the DNA released from bacterial cells after lysing with the developed antibacterial PPM columns is ready to be used by PCR with no further purification needed. Amplified DNA genes were detected by gel electrophoresis starting from small volume and cells concentration down to 10^2 CFU/ml. This makes it an attractive on-chip lysis device that can be used in sample preparation for bio-genetics and point-of-care diagnostics. The PPM columns were formed by photo-initiated free radical copolymerization of functional and cross-linker monomers with the assistance of porogenic solvents and photo-initiator. The porous network was synthesized directly inside a microfluidic channel fabricated in a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by well-controlled, high-throughput, and time and cost e effective laser micromachining. The unreacted double bonds at the surface of X-PMMA provide covalent bonding for the formation of the monolith, thus contributing to the mechanical stability of the PPM within the microchannel and eliminating the need for surface treatment. Two functional monomers belong to two different antibacterial polymers families have been used to form two antibacterial PPM columns. A functional monomer, N-(tert-butyloxycarbonyl)aminoethyl methacrylate, belongs to SMAMPs antibacterial polymer family was used to form antibacterial PPM column, SMAMPs-PPM. To demonstrate that cells were lysed via a dual mechanism, the active (functional) monomer, N-(tert-butyloxycarbonyl)aminoethyl methacrylate (Boc-AEMA), was protected by a Boc group to first demonstrate mechanical shearing lysis alone. Once the protecting group was removed the PPM became antibacterial, leading to improved performance. Both lysis mechanisms were thus validated. Furthermore, the lysis efficiency of the PPM was improved by tuning their hydrophobic-hydrophilic balance and determining the optimal flow rate, at which the bacterial cell walls were sufficiently mechanically sheared through the porous medium of the column to disrupt the cell membrane by physical contact with the antibacterial polymeric biocide covering the pore surface. To further con rm and validate the dual lysis mechanism with different monomer, an antibacterial PPM, DADMAC-PPM, from a functional monomer that is one of quaternary ammonium compounds, diallyldimethylammonium chloride (DAMMAC), which is intrinsically cationic and antibacterial, was developed. Also the e effect of the cross-linking monomer on bacterial growth inhibition, lysis efficiency and the mechanical stability of the PPM column within the microfluidic channel, using three different cross-linking monomers was studied. Moreover, the bonding e efficiency between two layers of X-PMMA substrate at different cross-linker contents was studied. Furthermore, the reusability of the QAC-PPM was investigated and compared with the previously developed SMAMPs-PPM. The cell lysis e efficiency of the biochips were characterized by qualitatively (semi-quantitatively) detecting DNA and quantitatively determining DNA concentration in the crude lysate collected at the outlet of the biochip. By using fluorometry, the ethidium bromide (EtBr) intercalation assay was utilized as an indicator of the presence of DNA in the cell lysate and the DNA concentration was determined by UV-Vis spectrophotometry. Furthermore, lysis was con rmed by o -chip PCR that was further analyzed by gel electrophoresis. The antibacterial PPM columns were reused for 20-30 lysis cycles without any evidence of physical damage to the monolith, significant performance degradation or DNA carryover when they were back-flushed between cycles

HEALING POTENCY OF HAEMATOCOCCUS PLUVIALIS EXTRACT FOR TREATING TYPE 2 DIABETES IN RATS

Objective: The present study aims to evaluate the antidiabetic effect of ethanolic extract of Haematococcus pluvialis (H. pluvialis) in streptozotocin (STZ)-induced diabetic rats.Methods: The antidiabetic activity of H. pluvialis was investigated by the determination of glucose and insulin levels, aspartate (AST), alanine transaminases (ALT), lipid profile including total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C) and high-density-lipoprotein-cholesterol (HDL-C). Histopathological examination of pancreas and liver were also carried out.Results: The results revealed that the levels of glucose, TC, TG, LDL-C as well as AST and ALT enzyme activities were increased significantly in diabetic rats. While, insulin and HDL-C levels decreased significantly in STZ-induced diabetic rats. The remediation of diabetic rats with H. pluvialis attenuated the elevated levels of glucose, TC, TG, LDL-C as well as AST and ALT activities in diabetic rats. Besides, it improved insulin, HDL-C levels, pancreas and hepatic architectures.Conclusion: H. pluvialis extract has a promising antidiabetic potency through attenuation of several metabolic disorders associated diabetes

ZnO hollow spheres arrayed molecularly-printed-polymer based selective electrochemical sensor for methyl-parathion pesticide detection

A highly sensitive electrochemical-based detector was fabricated to selectively sense methyl-parathion (MP). A Glassy carbon electrode (GCE) was functionalized with zinc oxide (ZnO) hollow spheres (ZnOHS) and a molecularly imprinted polymer (MIP) to form the developed sensor. Cyclic voltammetry (CV) was performed to synthesize a molecularly imprinted polymeric film on the ZnOHS modified GCE (GCE/ZnOHS) by electropolymerization of functional monomer, l-arginine (L-Arg), and template molecule, MP. The differential pulse voltammetry (DPV) was utilized to evaluate the efficiency of the electrochemical detection of MP under optimal conditions by the proposed sensor. The developed sensor recorded a good performance for detecting MP in the linear range of 5 × 10−9 to 0.1 × 10−4 mol L−1 (R2=0.985) with a detection limit (S/N = 3) of 0.5 × 10−9 mol L−1 and sensitivity of 571 nA/μmolL −1 cm −2. This electrochemical sensing system effectively detects MP in real samples with satisfactory recoveries of 90.4%, 91.9%, 118%, and 96.3% for fresh green beans, strawberry, tomato, and cabbage, respectively. © 2021 Elsevier B.V.1

MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN RAW CAPRINE MILK

ABSTRACT One hundred and fifty individual caprine milk samples were analyzed for Mycobacterium avium subsp. paratuberculosis (MAP). Out of 150 samples tested for MAP, 53 (35.33%) samples could be detected by Enzyme-Linked Immunosobent Assay (ELISA) technique. However, one (0.67%) sample was found positive in Polymerase Chain Reaction (PCR) method and failed to be isolated from all the examined samples

WITHDRAWN: Comparative study between bio-and phosphorus fertilization on growth, yield and fruit quality of banana (Musa spp.) grown on sandy soil

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