Multititration: The New Method for Implementing Ultrasensitive and Quantitative Multiplexed In-Field Immunoassays Despite Cross-Reactivity?

The accurate in-field titration of multiple pathogens is essential to efficiently describe and monitor environmental or biological contamination, isolate, act, and treat adequately. This underscores the requirement of portable, fast, quantitative, and multiplexed detection technologies, which, however, have not been properly developed so far, notably because it has been hindered by the phenomenon of cross-reactivity. In this work, we proposed a new analytical method based on the imaging through a portable device of lanthanide-based nanoparticles (YVO4:Eu) for spatially multiplexed detection, relying on a multiparameter analysis, i.e., a simultaneous analysis of all of the luminescence signals through the comparison to a calibration surface built in the presence of multiple analytes of interest. We then demonstrated the possibility to simultaneously quantify by multiplexed lateral flow assay (xLFA) the three enterotoxins SEG, SEH, and SEI in unknown mixtures, over two concentration decades (from a dozen of pg·mL-1 to few ng·mL-1). Assays were performed in less than an hour (25 min of strip migration followed by 30 min of drying at room temperature), the time during which the presence of the operator was not required for more than 5 min, in order to dip the strip and have it imaged by the reader. The concepts of nominal concentration recovery, coefficient of variation (CV), limit of blank (LOB), and limit of detection (LOD) were discussed in detail in the context of multiplexed assays. With our new definitions, quantitative results demonstrated a high recovery of the nominal concentrations (115%), reliability (CV = 20%), and sensitivity (LOBs of 3, 27, and 6 pg·mL-1 for SEG, SEH, and SEI respectively, and LODs of 6, 48, and 11 pg·mL-1 for SEG, SEH, and SEI, respectively). Based on this method, we observed an increase in sensitivity of 100 compared to the other multiplexed LFA labeled with gold particles and we approached the sensitivity of the simplex enzyme-linked immunosorbent assay (ELISA) performed with the same capture and detection antibodies. To conclude, our results, which are applicable to virtually any kind of multiplexed test, pave the way to the next generation of in-field analytical immunoassays by providing fast, quantitative, and highly sensitive multiplexed detection of biomarkers or pathogens.

Revealing the pulmonary surfactant corona on silica nanoparticles by cryo-transmission electron microscopy.

When inhaled, nanoparticles (NPs) deposit in alveoli and transit through the pulmonary surfactant (PS), a biofluid made of proteins and phospholipid vesicles. They form a corona reflecting the PS-nanomaterial interaction. Since the corona determines directly the NPs' biological fate, the question of its nature and structure is central. Here, we report on the corona architecture formed after incubation of positive or negative silica particles with Curosurf®, a biomimetic pulmonary surfactant of porcine origin. Using optical, electron and cryo-electron microscopy (cryo-TEM), we determine the pulmonary surfactant corona structure at different scales of observation. Contrary to common belief, the PS corona is not only constituted by phospholipid bilayers surrounding NPs but also by multiple hybrid structures derived from NP-vesicle interaction. Statistical analysis of cryo-TEM images provides interesting highlights about the nature of the corona depending on the particle charge. The influence of Curosurf® pre- or post-treatment is also investigated and demonstrates the need for protocol standardization.

Effect of Nanoparticles on the Bulk Shear Viscosity of a Lung Surfactant Fluid.

Inhaled nanoparticles (<100 nm) reaching the deep lung region first interact with the pulmonary surfactant, a thin lipid film lining the alveolar epithelium. To date, most biophysical studies have focused on particle-induced modifications of the film interfacial properties. In comparison, there is less work on the surfactant bulk properties and on their changes upon particle exposure. Here we study the viscoelastic properties of a biomimetic pulmonary surfactant in the presence of various engineered nanoparticles. The microrheology technique used is based on the remote actuation of micron-sized wires via the application of a rotating magnetic field and on time-lapse optical microscopy. It is found that particles strongly interacting with lipid vesicles, such as cationic silica (SiO2, 42 nm) and alumina (Al2O3, 40 nm) induce profound modifications of the surfactant flow properties, even at low concentrations. In particular, we find that silica causes fluidification, while alumina induces a liquid-to-soft solid transition. Both phenomena are described quantitatively and accounted for in the context of colloidal physics models. It is finally suggested that the structure and viscosity changes could impair the fluid reorganization and recirculation occurring during breathing.

Design and Applications of a Fluorescent Labeling Technique for Lipid and Surfactant Preformed Vesicles.

Amphiphilic molecules such as surfactants, lipids, and block copolymers can be assembled into bilayers and form vesicles. Fluorescent membrane labeling methods require the use of dye molecules that can be inserted into the bilayers at different stages of synthesis. To our knowledge, there is no generalized method for labeling preformed vesicles. Herein, we develop a versatile protocol that is suitable to both surfactant and lipid preformed vesicles and requires no separation or purification steps. On the basis of the lipophilic carbocyanine green dye PKH67, the methodology is assessed on zwitterionic phosphatidylcholine vesicles. To demonstrate its versatility, it is applied to dispersions of anionic or cationic vesicles, such as a drug administrated to premature infants with respiratory distress syndrome, or a vesicle formulation used as a fabric softener for home care applications. By means of fluorescence microscopy, we then visualize the interaction mechanisms of nanoparticles crossing live cell membranes and of surfactants adsorbed on a cotton fabric. These results highlight the advantages of a membrane labeling technique that is simple and applicable to a large number of soft matter systems.

Tailored stimuli-responsive interaction between particles adjusted by straightforward adsorption of mixed layers of Poly(lysine)-g-PEG and Poly(lysine)-g-PNIPAM on anionic beads.

We report a simple and versatile method to functionalize anionic colloid particles and control particle solubility. Poly(lysine)-based copolymers (PLL) grafted with polyethylene oxide (PLL-g-PEG) or poly(N-isopropylacrylamide) (PLL-g-PNIPAM) spontaneously adsorb on bare beads dispersed in aqueous solutions of the copolymers. The final composition of the mixed ad-layers formed (i.e. PEG/PNIPAM ratio) was adjusted by the polymer concentrations in solutions. While the (PLL-g-PEG)-coated particles were stable in a wide range of temperature, the presence of PLL-g-PNIPAM in the outer layer provided a reversible temperature-triggered aggregation at 32±1 °C. In the range of PNIPAM fraction going from 100% (beads fully covered by PLL-g-PNIPAM) down to a threshold 20% weight ratio (with 80% PLL-g-PEG), the particles aggregated rapidly to form micrometer size clusters. Below 20% weight fraction of PLL-g-PNIPAM, the kinetic was drastically lowered. Using PLL derivatives provides a straightforward route allowing to control the fraction of a functional chain (here PNIPAM) deposited on PEGylated particles, and in turn to adjust surface interaction and here the rate of particle-particle aggregation as a function of the density of functional chains. This approach can be generalized to many anionic surfaces onto which PLL is known to adhere tightly, such as glass or silica.