The paper describes a droplet-based microfluidic method for phenotypic-based antimicrobial susceptibility testing (AST). In particular, this micro-droplet-based phenotypic assay evaluates susceptibility of different bacterial strains towards antibiotics by tracking effects on individual bacterial cells, including changes in bacterial cell number and morphology. The platform was validated by applying the method to test the responses of E. coli ATCC 25922 and 6937 (a clinical isolate), in spiked urine samples at a concentration of 5 × 104 cfu mL?1, to the antibiotics ceftazidime and levofloxacin. Both E. coli strains showed dose-dependent inhibition of bacterial replication and morphological alteration. These correlated well with minimal inhibitory concentrations determined by the reference broth microdilution method. Discrete bacterial divisions and morphological changes were observed within 20 min of on-chip incubation, demonstrating performance of rapid AST directly on urine samples. As proof-of-concept, specific bead-based biosensors were tested for capture and detection of E. coli for on-bead proliferation. The method has the attractive feature of allowing the detection of at least one bacterium per bead in less than 30 min. It can potentially be used to isolate a specific bacterial strain directly from patient urine samples for AST monitoring.
Graphical Abstract (A) Schematic of the droplet microfluidic chip for bacterial detection and Antibiotic Susceptibility Testing (AST); (B) Time lapse proliferation images of green fluorescent protein expressing E. coli in droplets. (C) Bacterial proliferation on the bead-based sensor.
A lateral flow assay for rapid, simple and efficient determination of L. monocytogenes is presented. A monoclonal antibody (mAb) 1C1 against the peptide from P60 protein of L. monocytogenes was prepared and labeled with gold nanoparticles (AuNPs). The mAb 1C1 was paired with the mAb 10E7 against the P60 protein of all the Listeria spp. and used as a capture bioligand in a lateral flow assay. The AuNP-based strip test can detect the supernatant of eight common L. monocytogenes serotypes including 1/2a, 1/2b, and 4b with an equivalent detection limit of 3.7 × 106 CFU?mL?1 but does not detect four other Listeria spp. (L. ivanovii, L. innocua, L. welshimeri, and L. grayi). There was no cross-reactivity with six other Gram-negative and Gram-positive bacteria. The method was applied to the quantification of L. monocytogenes species in spiked milk samples within 13 h.
Graphical abstract Monoclonal antibody (mAb) 1C1 was prepared against the peptide from P60 protein of Listeria monocytogenes. The gold nanoparticle (AuNP) based strip sensor detects all the eight tested Listeria monocytogenes serotypes.
The authors describe three fluorescein-conjugated peptides generated by cell-phage display for use as a diagnostic probes for fluorescent detection and imaging of Salmonella enteritidis and Salmonella typhimurium. The authors also designed a polyvalent-directed peptide polymer synthesized with poly-D-lysine and bifunctional succinimidyl 3-(2-pyridyldithio)propionate with an affinity and sensitivity that is higher by more than an order of magnitude compared to single peptides due to multiple binding site interactions. In order to establish a diagnostic system for food poisoning, imaging analysis was performed using fluorescence microscopy. The limit of detection of the diagnostic system based on polyvalent directed peptide interaction is 102 colony-forming units per mL for Salmonella.
Graphical abstract Schematic of a fluorescent method for detection and imaging of Salmonella enteritidis and Salmonella typhimurium by using a fluorescein labeled polyvalent-directed peptide polymer (PDPP) with a high affinity and sensitivity as a diagnostic probe. The system uses a microplate reader and was applied to the detection of food poisoning.
An affinity-based protocol is described for the detection of Staphylococcus aureus (S. aureus). It is utilizing teicoplanin-functionalized magnetic beads as carriers. Teicoplanin, which binds to the walls of cells of S. aureus via five hydrogen bonds, acts as the recognition agent. Captured S. aureus is magnetically separated from the sample matrix and then specifically lysed by lysostaphin which cleaves the cross-linking pentaglycine bridges of peptidoglycan in the cell wall. Lastly, S. aureus is quantified via the inhibitory effect of released intracellular catalase on a chemiluminescent (CL) system composed of peroxidase, luminol, H2O2 and p-iodophenol because catalase decomposes H2O2. S. aureus can be detected with CL response in the 140 to 1.4?×?107 CFU·mL?1 concentration range and a detection limit as low as 47 CFU·mL?1 at a signal-to-noise ratio of 3. The method was evaluated by analyzing spiked samples including milk, human urine and saline injection solutions. The reliability was demonstrated by a recovery test and by comparison with a conventional plate counting method.
Graphical abstract An antibiotic-affinity protocol is developed to detect Staphylococcus aureus (S. aureus) by utilizing teicoplanin-functionalized magnetic beads (Teic-MBs) as carriers. S. aureus can be quantified by measuring the inhibition of luminol chemiluminescence (CL) signal by intracellular catalase.
The authors describe a method for amperometric determination of chloramine-T that is based on the indirect detection of chloramine-T by detecting p-quinone imine (p-QI) that is generated by oxidation of p-aminophenylboronic acid by chloramine-T. p-QI can be detected with excellent selectivity and at low potential by using a glassy carbon electrode. Hence, the method displays attractive features such as high sensitivity, wide detection range and excellent selectivity. The electrode has two linear responses in the 50 nM to 100 μM concentration range and a 6 nM detection limit. Compared to other electrochemical methods, this assay has a detection limit that is better by three orders of magnitude. The relative standard deviation is 3.4% for the determination of 10 μM of the medical chloramine-T sample, and the recovery of a samples containing chloramine-T at a level of 10 μM is 115%.
Graphical abstract Highly sensitive electrochemical detection of chloramine-T is achieved based on the reaction of chloramine-T with p-aminophenylboronic acid with a detection limit of 6 nM.
Porphyromonas gingivalis (P. gingivalis) is a pathogen causing periodontitis. A rapid assay is described for the diagnosis of periodontal infections related to P. gingivalis. The method is making use of gingipains, a group of P. gingivalis specific proteases as a detection biomarker. Magnetic-nanobeads were labeled with gingipain-specific peptide substrates and immobilized on a gold biosensing platform via gold-thiol linkage. As a result of this, the color of the gold layer turns black. Upon cleavage of the immobilized substrates by gingipains, the magnetic-nanobeads-peptide fragments were attracted by a magnet so that the golden surface color becomes visible again. This assay is highly sensitive and specific. It is capable of detecting as little as 49 CFU·mL?1 of P. gingivalis within 30 s. Examination of periodontitis patients and healthy control saliva samples showed the potential of the assay. The simplicity and rapidity of the assay makes it an effective point-of-care device.
Graphical abstract Schematic of the assay for the detection of P. gingivalis proteases as one of the promising biomarkers associated with periodontal diseases.
Light-activated antimicrobial coatings were obtained by the covalently immobilizing photo-sensitizers in a hybrid organic/inorganic matrix. These coatings were deposited via sol-gel chemistry using epoxy and methyl functional silanes. The light-activated chromophores used in this study were Methylene Blue, Toluidine Blue O, and Rose Bengal. The immobilized photo-sensitizers did not leach from the coatings. The mechanically durable hybrid coatings comprising 2.5% by weight of Rose Bengal had a good adhesion to the glass surface. These coatings were tested for the photo-deactivation of Escherichia coli and Staphylococcus aureus using illumination by a commercial fluorescent lamp. Log reduction of E. coli and S. aureus were >4 when illuminated by the fluorescent lamp in 1 and 3?h, respectively. Due to its high mechanical durability and chemical resistance, such light-activated hybrid coatings are promising candidates for indoor applications in healthcare facilities.
A carbon paste electrode (CPE) was modified with Pt-Co nanoparticles and 2-(3,4-dihydroxyphenethyl)isoindoline-1,3-dione (3,4-DHPID) and then used for determination of N-acetylcysteine (N-AC) in the presence of paracetamol (PC) and folic acid (FA). The Pt-Co nanoparticles were synthesized by the polyol method and characterized by X-ray diffraction, energy dispersive X-ray analysis and transmission electron microscopy. The modified CPE displays good electrocatalytic activity towards the electrooxidation of N-AC in solution of pH 7.0. It was applied to the determination of N-AC in the presence of PC and FA (with well separated signals peaking at 0.2, 0.55 and 0.86 V vs. Ag/AgCl) by using square wave voltammetry. The peak currents are linearly dependent on the concentrations of N-AC, PC and FA in the respective ranges from 0.07 to 500, 1.0 to 850, and 2.0 to 550 μmol·L?1, with detection limits of 0.009, 0.6 and 0.8 μmol·L?1. The modified CPE was applied to the determination of N-AC, PC and FA in (spiked) pharmaceutical and biological samples.
Graphical abstract Pt-Co nanoparticles and 2-(3,4-dihydroxyphenethyl)isoindoline-1,3-dione were used for modification of a carbon paste electrode which then was used for sensitive determination of N-actylcysteine in the presence of paracetamol and folic acid.
An F0F1-ATPase-based aptasensor is described for the fluorometric determination of Vibrio parahaemolyticus. Chromatophores containing F0F1-ATPases were first prepared from Rhodospirillum rubrum cells. Then, an aptamer-functionalized chromatophore acts as the capture probe, and a chromatophore labeled with the pH probe fluorescein acts as the signalling probe. In the presence of V. parahaemolyticus, the rotation rate of F0F1-ATPase is decreased due to the formation of the aptamer-chromatophore complex. This leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and the fluorescence of the pH probe F1300 is accordingly decreased. The relative fluorescence varies linearly over the 15 to 1.5?×?106 cfu·mL?1Vibrio parahaemolyticus concentration range, and the limit of detection is 15 cfu·mL?1. The method was applied to analyze artificially contaminated salmon samples where it showed excellent perfomance.
Graphical abstract In this assay, aptamer functionalized chromatophores act as a capture probe, and the fluoresce in labeled chromatophores as signalling probe. The formation of aptamer-chromatophore complex leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and fluorescence intensity is accordingly decreased.
In this study, the effective TiO2/Ag composite antibacterial aerogel powder is prepared by facile sol–gel method and ethanol supercritical technology. The surface morphology, structural properties, and chemical components are monitored by scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), and energy disperse?spectroscopy (EDS). Meanwhile, absorbance spectra and specific surface area of TiO2/Ag composite aerogel are characterized by UV-Vis spectra and Brunauer–Emmett–Teller. The TiO2/Ag composite aerogel with Ti/Ag molar ratios of 10:1, 30:1, 50:1 are measured for its antibacterial property by using Escherichia coliform (E.coli) and Staphylococcus aureus (S. aureus). The results show that the size of TiO2 and Ag nanoparticles are 40?nm and 25?nm, respectively. Simultaneously, the obtained composite aerogel with a porous structure possessed a surface area of 148?m2/g, an average pore size 11.5?nm, and a pore volume 0.39?cm3/g. With the increase of Ag content, the antibacterial properties of composite aerogel are greatly improved compared with pure TiO2 aerogel. When Ag/Ti molar ratios was 1:10, the highest antibacterial rate can up to 99%, and the inhibition bands of E. coli and S. aureus are 23?mm and 19?mm, respectively.
The authors describe a sensitive surface-enhanced Raman scattering (SERS)-based aptasensor for the detection of the food pathogen Vibrio parahaemolyticus. Nanostructures consisting of Fe3O4@Au particles wrapped with graphene oxide (GO) were used both as SERS substrates and separation tools. A first aptamer (apt 1) was immobilized on the Fe3O4@Au/GO nanostructures to act as a capture probe via the affinity binding of aptamer and V. parahaemolyticus. A second aptamer (apt-2) was modified with the Raman reporter molecule TAMRA to act as a SERS sensing probes that binds to the target the same way as the Fe3O4@Au/GO-apt 1. The sandwich formed between Fe3O4@Au/GO-apt 1/V. parahaemolyticus and apt 2-TAMRA can be separated with the aid of a magnet. The concentration of V. parahaemolyticus can be quantified by measurement of the SERS intensity of TAMRA. Under optimal conditions, the signal is linearly related to the V. parahaemolyticus concentration in the range between 1.4 × 102 to 1.4 × 106 cfu·mL?1, with a detection limit of 14 cfu·mL?1. Recoveries ranging from 98.5% to 105% are found when analyzing spiked salmon samples. In our perception, the assay described here is a useful tool for quantitation of V. parahaemolyticus in real samples.
Graphical abstract GO wrapped Fe3O4@Au nanostructures were synthesized as the substrate and modified with with a first aptamer (apt 1) to capture V. parahaemolyticus. TAMRA labelled aptamer 2 was then used as signal probe. The V. parahaemolyticus concentrations are closely related to the Raman intensity of TAMRA.
We have previously shown that liquid extraction surface analysis (LESA) mass spectrometry (MS) is a technique suitable for the top-down analysis of proteins directly from intact colonies of the Gram-negative bacterium Escherichia coli K-12. Here we extend the application of LESA MS to Gram-negative Pseudomonas aeruginosa PS1054 and Gram-positive Staphylococcus aureus MSSA476, as well as two strains of E. coli (K-12 and BL21 mCherry) and an unknown species of Staphylococcus. Moreover, we demonstrate the discrimination between three species of Gram-positive Streptococcus (Streptococcus pneumoniae D39, and the viridans group Streptococcus oralis ATCC 35037 and Streptococcus gordonii ATCC35105), a recognized challenge for matrix-assisted laser desorption ionization time-of-flight MS. A range of the proteins detected were selected for top-down LESA MS/MS. Thirty-nine proteins were identified by top-down LESA MS/MS, including 16 proteins that have not previously been observed by any other technique. The potential of LESA MS for classification and characterization of novel species is illustrated by the de novo sequencing of a new protein from the unknown species of Staphylococcus.
Flower-like ceria (CeO2) architectures consisting of well aligned nanosheets were first synthesized by a glycol solvothermal method. The size of CeO2 architectures is about 5?μm in width and 10?μm in length, with the nanosheets thickness below 100?nm. Subsequently, the adsorbed Ag ions on the surface of CeO2 were in situ reduced to form Ag nanoparticles (NPs), leading to the fabrication of Ag/CeO2 hybrid architectures (HAs). The formed Ag NPs with sizes of 20–40?nm were uniformly loaded on the surface of the CeO2 sheets. The antibacterial properties of Ag/CeO2 HAs against Gram-negative E. coli and Gram-positive S. aureus were evaluated by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and a filter paper inhibition zone method. The results demonstrated that Ag/CeO2 HAs displayed excellent antibacterial activity toward S. aureus and E. coli, which were attributed to the synergistic antibacterial effect between Ag NPs and CeO2 in HAs. Here, CeO2 nanoflowers as a new substrate could restrict Ag NPs aggregations and improve their antibacterial activities. Therefore, the resulted Ag/CeO2 HAs would be considered as a promising antibacterial agent.
An ultrasensitive fluorometric assay is described for the determination of the activity of the enzyme α-glucosidase in waters and living cells. Carbon dots doped with nitrogen and boron (N,B-CDs) were prepared that have excitation/emission peaks at 400/510 nm and a fluorescence quantum yield of 47%. 4-Nitrophenylglucoside is added and then hydrolyzed by α-glucosidase to form yellow 4-nitrophenol which screens off fluorescence due to an inner filter effect. The method was applied to the determination of α-glucosidase activity and has a 3 mU mL?1 detection limit. It was subsequently applied to the determination of the α-glucosidase inhibitor acarbose which can be determined in a concentration as low as 10 nM (at three times the standard deviation versus slope). The method was also applied to the determination of α-glucosidase activity and acarbose in living HeLa cells and MCF-7 cells. The method is simple, sensitive, and excellently selective.
An ion of m/z 110.06036 (ion formula [C6H8NO]+; error: 0.32 mDa) was observed in the collision induced dissociation tandem mass spectrometry experiments of protonated N-(3-aminophenyl)benzamide, which is a rearrangement product ion purportedly through nitrogen-oxygen (N–O) exchange. The N–O exchange rearrangement was confirmed by the MS/MS spectrum of protonated N-(3-aminophenyl)-O18-benzamide, where the rearranged ion, [C6H8NO18]+ of m/z 112 was available because of the presence of O18. Theoretical calculations using Density Functional Theory (DFT) at B3LYP/6-31 g(d) level suggest that an ion-neutral complex containing a water molecule and a nitrilium ion was formed via a transition state (TS-1), followed by the water molecule migrating to the anilide ring, eventually leading to the formation of the rearranged ion of m/z 110. The rearrangement can be generalized to other protonated amide compounds with electron-donating groups at the meta position, such as, –OH, –CH3, –OCH3, –NH(CH3)2, –NH-Ph, and –NHCOCH3, all of which show the corresponding rearranged ions in MS/MS spectra. However, the protonated amide compounds containing electron-withdrawing groups, including –Cl, –Br, –CN, –NO2, and –CF3, at the meta position did not display this type of rearrangement during dissociation. Additionally, effects of various acyl groups on the rearrangement were investigated. It was found that the rearrangement can be enhanced by substitution on the ring of the benzoyl with electron-withdrawing groups.
We have prepared an environmental friendly sorbent by modifying multi-walled carbon nanotubes with tannic acid. The adsorption of La (III), Tb (III) and Lu (III) as a function of contact time, initial solution pH, and quantity of adsorbent was studied using a batch technique. Both Langmuir and Freundlich isotherms can be used to describe the process. The major adsorption mechanisms were attributed to ion exchange and surface complexation. The kinetics of the adsorption follows a pseudo-second-order model. The thermodynamic functions ΔH, ΔG, and ΔS indicate that the sorption is endothermically driven. The adsorbed ions can be readily desorbed from the surface with 1 M hydrochloric acid.
Figure An environmental friendly sorbent, multi-walled carbon nanotubes modified with tannic acid has been prepared and used for the adsorption of REEs. Adsorption capacity, Langmuir and Freundlich adsorption isotherms, kinetics, and thermodynamic functions have been investigated. The major adsorption mechanism can be attributed to ion exchange and surface complexation.
The authors report an aptaelectrode based on graphene modified iron-oxide chitosan hybrid (CHIT-IO-GR) nanocomposite film deposited on fluorine tin oxide (FTO) for the detection of the Mycobacterium tuberculosis specific antigen MPT64. The biotinylated DNA aptamer sequence specific to the MPT64 was immobilized onto the CHIT-IO-GR/FTO electrode by using streptavidin-biotin interactions. XRD, FT-IR, FE-SEM and electrochemical studies were applied to monitor the steps of the fabrication. The aptaelectrode, operated best at typical voltage of 0.44 V, exhibited a limit of detection (LOD) of 0.9 fg?mL?1 within 20 min. The biosensor retained about 80% of its initial activity after 10 uses. The potential application of the aptasensor was established by spike-in studies to obtain recoveries between 83 and 95%.
Graphical abstract An electrochemical aptaelectrode based on nanocomposite consisting of chitosan (CHIT), iron-oxide nanoparticles (IO) and functionalized graphene (GR) has been fabricated to detect M. tb antigen MPT64 with an LOD of 0.9 fg?mL?1 within 20 min.
With the help of Tn5 transposon technique, gene yfjB encoding NAD kinase in Escherichia coli (E. coli) was inserted into chromosome of recombinant E. coli polyhydroxybutyrate (PHB) containing PHB synthesis operon integrated in the host genome. After successful transposition of an extra yfjB gene copy into genome, the selected recombinant named E. coli PHBTY4 showed stronger NAD kinase activity than that of E. coli PHB. Shake flask studies suggested that both cell dry weight and PHB accumulation were significantly increased in E. coli PHBTY4 compared with that of the control. E. coli PHBTY4 produced approximately 23 g/L PHB compared with its control which synthesized only 10 g/L PHB when grown under the same conditions in a 6 L fermentor after 32 h of cultivation. In addition, E. coli PHBTY4 maintained high genetic stability during the cultivation processes. These results revealed a practical method to construct genetically stable strains harboring extra NAD kinase gene to enhance NADP(H)-dependent bio-reactions. 相似文献
Species of genus Burkholderia display different interaction profiles in the environment, causing either several diseases in plants and animals or being beneficial to some plants, promoting their growth, and suppressing phytopathogens. Burkholderia spp. also produce many types of biomolecules with antimicrobial activity, which may be commercially used to protect crops of economic interest, mainly against fungal diseases. Herein we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate secondary metabolites produced by B. seminalis TC3.4.2R3 in monoculture and coculture with plant pathogen Fusarium oxysporum. The siderophore pyochelin and the rhamnolipid Rha-Rha-C15-C14 were detected in wild-type B. seminalis strain, and their productions were found to vary in mutant strains carrying disruptions in gene clusters associated with antimicrobial compounds. Two mycotoxins were detected in F. oxysporum. During coculture with B. seminalis, metabolites probably related to defense mechanisms of these microorganisms were observed in the interspecies interaction zone. Our findings demonstrate the effective application of MALDI-MSI in the detection of bioactive molecules involved in the defense mechanism of B. seminalis, and these findings suggest the potential use of this bacterium in the biocontrol of plant diseases caused by F. oxysporum.
Single-run mass spectrometry has enabled the detection and quantifications of E. coli proteins. A total of 2068 proteins quantified by intensity-based absolute quantification (iBAQ) Schwanhäusser et al.: (Nature. 473, 337–342, 2011) procedure were obtained with single enzyme-trypsin, without pre-fractionation, by quadruplicate long liquid chromatography runs coupled with high-resolution linear trap quadrupole (LTQ)-Orbitrap Velos mass spectrometry. The single-run of 12 h has ability to cover almost 98% of the quadruplicate LC-MS/MS runs of E. coli proteome and is therefore almost equivalent to quadruplicate LC-MS/MS runs. These quantified proteins are about 52% of the total proteins present in E. coli genome according to Uniprot database. The quantified proteins covered almost all of the proteins in folate biosynthesis. Remarkably greater part of Gene Ontology (GO) Barrell et al.: (Nucleic Acids Res. 37, D396–D403, 2009), Ashburner et al.: (Nat. Genet. 25, 25–29, 2000) annotations, signaling pathways along with protein-protein interactions were covered. Some of the important biological processes-cell cycle, DNA repair, ion transport, ubiquinone biosynthetic process, pseudouridine synthesis, peptidoglycan biosynthetic process, RNA processing, and translation-revealed protein-protein interaction network generated by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) Jensen, et al.:(Nucleic Acids Res 37, D412-D126, 2009) database. Therefore, to achieve the saturation point of detection of maximum number of proteins in single LC-MS/MS run, 12-h liquid chromatography gradient is appropriate.
