Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

An emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

Adsorption mechanism of bacteria onto a Na-montmorillonite surface with organic and inorganic calcium

The adsorption of bacteria onto the Na-montmorillonite (Na-MMT) was studied as a function of time, bacterial concentration, temperature and pH with the introduction of the organic and inorganic calcium sources. The results indicated that albeit revealing the same adsorption mechanism, the organic calcium (i.e., Ca(CH3COO)2) proposed in this study is more beneficial and environmentally friendly than the inorganic calcium (i.e., CaCl2) in terms of the adsorption of bacteria onto the Na-MMT surface, which can be ascribed to the formation of the denser aggregates in the Na-MMT with Ca(CH3COO)2. Meanwhile, the adsorption kinetics and isotherms followed the pseudo-second-order kinetic model and Langmuir Equation for both two calcium sources. Meanwhile, the adsorption bands of the water molecules on the minerals were observed to shift significantly after the bacterial adsorption, showing that the hydrogen bonding on the Na-MMT surface played an important role during this process. A value of ΔH0 > 0 indicated that the bacterial adsorption was affected by van der Waals force and hydrophobic interaction. Finally, the negative zeta potentials of the Na-MMT increased with the addition of Ca2+ ions, and the experimental data also showed that the adsorption of bacteria onto the Na-MMT was mainly determined by the electrostatic and non-electrostatic forces.

Enhanced Hydrophilic and Electrophilic Properties of Polyvinyl Chloride (PVC) Biofilm Carrier

Polyvinyl chloride (PVC) biofilm carrier is used as a carrier for bacterial adsorption in wastewater treatment. The hydrophilicity and electrophilicity of its surface play an important role in the adsorption of bacteria. The PVC biofilm carrier was prepared by extruder, and its surface properties were investigated. In order to improve the hydrophilicity and electrophilic properties of the PVC biofilm carrier, polyvinyl alcohol (PVA) and cationic polyacrylamide (cPAM) were incorporated into polyvinyl chloride (PVC) by blending. Besides, the surface area of the PVC biofilm carrier was increased by azodicarbonamide modified with 10% by weight of zinc oxide (mAC). The surface contact angle of PVC applied by PVA and cPAM at 5 wt %, 15 wt % was 81.6°, which was 18.0% lower than pure PVC. It shows the significant improvement of the hydrophilicity of PVC. The zeta potential of pure PVC was −9.59 mV, while the modified PVC was 14.6 mV, which proves that the surface charge of PVC changed from negative to positive. Positive charge is more conducive to the adsorption of bacteria. It is obvious from the scanning electron microscope (SEM) images that holes appeared on the surface of the PVC biofilm carrier after adding mAC, which indicates the increase of PVC surface area.

Development of Poly(2-Methacryloyloxyethyl Phosphorylcholine)-Functionalized Hydrogels for Reducing Protein and Bacterial Adsorption

A series of hydrogels with intrinsic antifouling properties was prepared via surface-functionalization of poly(2-hydroxyethyl methacrylate) [p(HEMA)]-based hydrogels with the biomembrane-mimicking zwitterionic polymer, poly(2-methacryloyloxyethyl phosphorylcholine) [p(MPC)]. The p(MPC)-modified hydrogels have enhanced surface wettability, high water content retention (61.0%–68.3%), and good transmittance (>90%). Notably, the presence of zwitterionic MPC moieties at the hydrogel surfaces lowered the adsorption of proteins such as lysozyme and bovine serum albumin (BSA) by 73%–74% and 59%–66%, respectively, and reduced bacterial adsorption by approximately 10%–73% relative to the unmodified control. The anti-biofouling properties of the p(MPC)-functionalized hydrogels are largely attributed to the dense hydration layer formed at the hydrogel surfaces by the zwitterionic moieties. Overall, the results demonstrate that biocompatible and antifouling hydrogels based on p(HEMA)-p(MPC) structures have promising potential for application in biomedical materials.

Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-L-lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane.

Effect of Acidithiobacillus ferrooxidans on Humic-Acid Passivation Layer on Pyrite Surface

The effect of Acidithiobacillus ferrooxidans on the humic-acid passivation layer on pyrite surfaces was studied by atomic-force microscopy, leaching experiments, and adsorption experiments. Atomic-force-microscopy results showed that humic-acid was adsorbed onto the pyrite surface. The bacteria grew and reproduced on the humic-acid layer. Leaching experiments showed that the humic-acid passivation layer prevented the oxidation of pyrite by Fe3+ under aseptic conditions. Bacteria destroyed the humic-acid layer, promoted pyrite oxidation, and increased the oxidation of pyrite from 1.64% to 67.9%. Bacterial adsorption experiments showed that the humic-acid passivation layer decreased the speed of bacterial adsorption on the pyrite surface but had no effect on the number of bacteria adsorbed on the pyrite surface. The maximum number of bacteria adsorbed by pyrite with and without the humic-acid layer was 4.17 × 1010 cells∙mL−1 and 4.4 × 1010 cells∙mL−1, respectively. Extracellular polymeric stratum layer of bacteria cultured at different concentrations of humic-acid was extracted and analyzed. This layer could destroy the humic-acid layer and promote pyrite oxidation.