The variation in the extracellular metabolites of RAW 264.7 cells obtained from different passage numbers (passage 9, 12 and 14) was examined. The impact of different harvesting protocols (trypsinization and scraping) on recovery of intracellular metabolites was then assessed. The similarity and variation in the cell metabolome was investigated using 1H NMR metabolic profiling modeled using multivariate data analysis. The characterization and quantification of metabolites was performed to determine the passage-related and harvesting-dependent effects on impacted metabolic networks. The trypsinized RAW cells from lower passages gave higher intensities of most identified metabolites, including asparagine, serine and tryptophan. Principal component analysis revealed variation between cells from different passages and harvesting methods, as indicated by the formation of clusters in score plot. Analysis of S-plots revealed metabolites that acted as biomarkers in discriminating cells from different passages including acetate, serine, lactate and choline. Meanwhile lactate, glutamine and pyruvate served as biomarkers for differentiating trypsinized and scraped cells. In passage-dependent effects, glycolysis and TCA cycle were influential, whereas glycerophospholipid metabolism was affected by the harvesting method. Overall, it is proposed that typsinized RAW cells from lower passage numbers are more appropriate when conducting experiments related to NMR metabolomics.
In the present study, a nanocomposite of f-MWCNTs-chitosan-Co was prepared by the immobilization of Co(II) on f-MWCNTs-chitosan by a self-assembly method and used for the quantitative determination of paracetamol (PR). The composite was characterized by field emission scanning electron microscopy (FESEM) and energy dispersive x-ray analysis (EDX). The electroactivity of cobalt immobilized on f-MWCNTs-chitosan was assessed during the electro-oxidation of paracetamol. The prepared GCE modified f-MWCNTs/CTS-Co showed strong electrocatalytic activity towards the oxidation of PR. The electrochemical performances were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Under favorable experimental conditions, differential pulse voltammetry showed a linear dynamic range between 0.1 and 400 μmol L-1 with a detection limit of 0.01 μmol L-1 for the PR solution. The fabricated sensor exhibited significant selectivity towards PR detection. The fabricated sensor was successfully applied for the determination of PR in commercial tablets and human serum sample.
Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.
Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based high-throughput and inexpensive method for tracking bacterial adherence and internalization through flow cytometry to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and cell-dependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study host-bacteria interaction.
Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)-TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006-2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.
Rapid detection of foodborne pathogens is crucial as ingestion of contaminated food products may endanger human health. Thus, the objective of this study was to develop a biosensor using reduced graphene oxide-carbon nanotubes (rGO-CNT) nanocomposite via the hydrothermal method for accurate and rapid label-free electrochemical detection of pathogenic bacteria such as Salmonella enterica. The rGO-CNT nanocomposite was characterized using Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffraction and transmission electron microscopy. The nanocomposite was dropped cast on the glassy carbon electrode and further modified with amino-modified DNA aptamer. The resultant ssDNA/rGO-CNT/GCE aptasensor was then used to detect bacteria by using differential pulse voltammetry (DPV) technique. Synergistic effects of aptasensor was evident through the combination of enhanced electrical properties and facile chemical functionality of both rGO and CNT for the stable interface. Under optimal experimental conditions, the aptasensor could detect S. Typhimurium in a wide linear dynamic range from 101 until 108 cfu mL-1 with a 101 cfu mL-1 of the limit of detection. This aptasensor also showed good sensitivity, selectivity and specificity for the detection of microorganisms. Furthermore, we have successfully applied the aptasensor for S. Typhimurium detection in real food samples.
The identification of rice bacterial leaf blight disease requires a simple, rapid, highly sensitive, and quantitative approach that can be applied as an early detection monitoring tool in rice health. This paper highlights the development of a turn-off fluorescence-based immunoassay for the early detection of Xanthomonas oryzae pv. oryzae (Xoo), a gram-negative bacterium that causes rice bacterial leaf blight disease. Antibodies against Xoo bacterial cells were produced as specific bio-recognition molecules and the conjugation of these antibodies with graphene quantum dots and gold nanoparticles was performed and characterized, respectively. The combination of both these bio-probes as a fluorescent donor and metal quencher led to changes in the fluorescence signal. The immunoreaction between AntiXoo-GQDs, Xoo cells, and AntiXoo-AuNPs in the immuno-aggregation complex led to the energy transfer in the turn-off fluorescence-based quenching system. The change in fluorescence intensity was proportional to the logarithm of Xoo cells in the range of 100-105 CFU mL-1. The limit of detection was achieved at 22 CFU mL-1 and the specificity test against other plant disease pathogens showed high specificity towards Xoo. The detection of Xoo in real plant samples was also performed in this study and demonstrated satisfactory results.
An optical biosensor based on glutamate dehydrogenase (GLDH) immobilized in a chitosan film for the determination of ammonium in water samples is described. The biosensor film was deposited on a glass slide via a spin-coating method. The ammonium was measured based on beta-nicotinamide adenine dinucleotide (NADH) oxidation in the presence of alpha-ketoglutaric acid at a wavelength of 340 nm. The biosensor showed optimum activity at pH 8. The optimum chitosan concentrations and enzyme loading were found to be at 2% (w/v) and 0.08 mg, respectively. Optimum concentrations of NADH and alpha-ketoglutaric acid both were obtained at 0.15 mM. A linear response of the biosensor was obtained in the ammonium concentration range of 0.005 to 0.5 mM with a detection limit of 0.005 mM. The reproducibility of the biosensor was good, with an observed relative standard deviation of 5.9% (n=8). The biosensor was found to be stable for at least 1 month when stored dry at 4 degrees C.
The phage N15 protelomerase enzyme (TelN) is essential for the replication of its genome by resolution of its telRL domain, located within a telomerase occupancy site (tos), into hairpin telomeres. Isolation of TelN for in vitro processing of tos, however, is a highly complex process, requiring multiple purification steps. In this study a simplified protocol for crude total protein extraction is described that retains the tos-cleaving activity of TelN for at least 4 weeks, greatly simplifying in vitro testing of its activity. This protocol may be extended for functional analysis of other phage and bacterial proteins, particularly DNA-processing enzymes.
Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.
We compared four brands of microtubes with respect to their suitability for long-range polymerase chain reactions (PCRs). One of the four brands was found to have an inhibitory effect, decreasing PCR yields. The effect was universal across different PCR or enzyme systems. Increased ultraviolet absorbance suggests leaching of unknown chemical species into PCR mixtures. However, this could not be confirmed by high-performance liquid chromatography-mass spectrometry analysis. Nevertheless, our article demonstrates a clear impact of the choice of microtubes on long-range PCR success. Due consideration should be given to the PCR microtubes when determining optimal reaction conditions for long-range PCR.
Following hematopoietic stem cell transplantation (HSCT), it is important to determine whether engraftment is successful and to track the dynamic changes of the graft. Tandem repeats such as minisatellites and microsatellites are currently the most established markers for chimerism application. We have developed a reliable method to quantitatively evaluate engraftment status in post-allogeneic HSCT patients using variable number of tandem repeat (VNTR) markers and "lab-on-a-chip" microfluidic electrophoresis technology. Following identification of an informative marker by conventional polymerase chain reaction (PCR), donor chimerism percentage was calculated based on a standard curve generated from artificially mixed patient-donor DNA-specific alleles in serial dilutions. All PCR products were mixed with commercial gel dye and loaded into Agilent DNA 1000 microfluidic LabChips for DNA sizing and quantitation. In 44 patients, separation of pretransplant and donor DNA fragments was resolved clearly and accomplished rapidly within 30min. Chimerism analysis using this platform is able to detect an amount as low as 6.3% donor DNA with acceptable coefficient of variation values. We also demonstrated concordant chimerism analysis findings using both microchip tandem repeats and real-time PCR quantitation of insertion-deletion polymorphisms. This microchip platform obviates the need for fluorescently labeled primers or any post-PCR sample manipulation. Quantitative monitoring of post-HSCT chimerism status using microfluidic electrophoresis is a useful tool for both large- and small-scale post-HSCT chimerism centers.
In this study, an amino-modified aptasensor using multi-walled carbon nanotubes (MWCNTs)-deposited ITO electrode was prepared and evaluated for the detection of pathogenic Salmonella bacteria. An amino-modified aptamer (ssDNA) which binds selectively to whole-cell Salmonella was immobilised on the COOH-rich MWCNTs to produce the ssDNA/MWCNT/ITO electrode. The morphology of the MWCNT before and after interaction with the aptamers were observed using scanning electron microscopy (SEM). Cyclic voltammetry and electrochemical impedance spectroscopy techniques were used to investigate the electrochemical properties and conductivity of the aptasensor. The results showed that the impedance measured at the ssDNA/MWCNT/ITO electrode surface increased after exposure to Salmonella cells, which indicated successful binding of Salmonella on the aptamer-functionalised surface. The developed ssDNA/MWCNT/ITO aptasensor was stable and maintained linearity when the scan rate was increased from 10 mV s-1 to 90 mV s-1. The detection limit of the ssDNA/MWCNT/ITO aptasensor, determined from the sensitivity analysis, was found to be 5.5 × 101 cfu mL-1 and 6.7 × 101 cfu mL-1 for S. Enteritidis and S. Typhimurium, respectively. The specificity test demonstrated that Salmonella bound specifically to the ssDNA/MWCNT/ITO aptasensor surface, when compared with non-Salmonella spp. The prepared aptasensor was successfully applied for the detection of Salmonella in food samples.
An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC50 of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.
A method using solid phase extraction and liquid chromatography-tandem mass spectrometry to quantitatively detect mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine samples was developed and validated. The relevant metabolites were identified using multiple reaction monitoring in positive ionization mode using nalorphine as an internal standard. The method was validated for accuracy, precision, recovery, linearity, and lower limit of quantitation. The intra- and inter-day accuracy and precision were found in the range of 83.6-117.5% with coefficient of variation less than 13%. The percentage of recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine was within the range of 80.1-118.9%. The lower limit of quantification was 1 ng/mL for mitragynine, 2 ng/mL for 16-carboxy mitragynine, and 50 ng/mL for 9-O-demethyl mitragynine. The developed method was reproducible, high precision and accuracy with good linearity and recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine.
DNA-templated silver nanocluster (AgNC), a new promising fluorescence probe has gained importance in biosensing and bioimaging in recent years. We employed a label-free AgNC to detect an intracellular transcription factor known as forkhead box p3 (FOXP3), which is the master regulator of regulatory T cells (Tregs) suppressive function. We developed an optimized method for the detection of messenger ribonucleic acid (mRNA) of FOXP3 by hybridizing AgNC and G-rich to the target FOXP3 mRNA of a MCF-7 cells. MCF-7 cells are chosen as a model as it readily expresses FOXP3. The hybridized samples were examined with UV illuminator and further verified with fluorescence spectroscopy, fluorescence microscope and flow cytometry. The successful hybridization of a three-way junction with AgNC, G-rich and mRNA FOXP3 target generated an improved fluorescence intensity with a spectral shift. We have successfully delivered the green fluorescing AgNC and G-rich into MCF-7 cells, producing a shift to red fluorescing cells corroborated by flow cytometry results. In summary, our approach enables the detection of intracellular FOXP3 nucleic acid and holds considerable potential in establishing a non-lethal intracellular detection system which would be crucial for the isolation of regulatory T-cells (Tregs) when combined with other cell surface markers.
Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant β2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two β2-agonists were; IC50 salbutamol = ∼0.310 μg/ml, IC50 clenbuterol = ∼0.076 μg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 μg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 μg/ml currently implemented by WADA and may be of potential use in human doping urinalysis.
Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.
Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity.
The quality of a nucleotide-based library such as a synthetic antibody library is highly dependent on the diversity available. Diversity can be generated using degenerate oligonucleotides introduced during gene assembly. Conventional approaches to gene assembly are not efficient for oligonucleotides with long stretches of degeneracy. We propose an efficient alternative for simultaneous introduction of three randomized regions in a synthetic antibody gene via temperature cascading. The strategy takes advantage of DNA reannealing kinetics. The strategy can be adopted for generating diversity of gene inserts during the construction of nucleotide-based libraries.