Uncovering toxicological complexity by multi-dimensional screenings in microsegmented flow: modulation of antibiotic interference by nanoparticles

Abstract

The technique of microsegmented flow was applied for the generation of two- and higher dimensional concentration spaces for the screening of toxic effects of selected substances on the bacterium Escherichia coli at the nanolitre scale. Up to about 5000 distinct experiments with different combinations of effector-concentrations could be realized in a single experimental run. This was done with the help of a computer program controlling the flow rates of effector-containing syringe pumps and resulted in the formation of multi-dimensional concentration spaces in segment sequences. Prior to the application of this technique for toxicological studies on E. coli the accuracy of this method was tested by simulation experiments with up to five dissolved dyes with different spectral properties. Photometric microflow-through measurement of dye distribution inside the concentration spaces allowed the monitoring of microfluid segment compositions. Finally, we used this technique for the investigation of interferences of the antibiotics ampicillin and chloramphenicol towards E. coli cultures and their modulation by silver nanoparticles by measuring bacterial autofluorescence. Each concentration point in this three-dimensional concentration space was represented by 4 or 5 single segments. Thus, a high reliability of the measured dose/response relations was achieved. As a result, a complex response pattern was discovered including synergistic and compensatory effects as well as the modulation of the range of stimulation of bacterial growth by a sublethal dose of chloramphenicol by silver nanoparticles.

Investigation of mixture toxicity of widely used drugs caffeine and ampicillin in the presence of an ACE inhibitor on bacterial growth using droplet-based microfluidic technique

“…Details on the fluidic experimental setup, the optical microdevices, and the applied methods for the realization and analysis of three-dimensional concentration spaces have been described in an earlier publication [4]. Hereby, a similar experimental setup was used for oneup to three-dimensional screening of effectors based on a syringe pump system with six dosing units (Cetoni GmbH, Korbußen, Germany) ( Figure 1).…”

“…For precultivation, a single colony of E. coli was removed from the LB (Lysogeny broth) agar plate (10 g/l bacterial peptone, 5 g/l yeast extract, 5 g/l NaCl, and 13 g/l agar), transferred into an Erlenmeyer flask filled with 10 ml synthetic medium (0.56 g K 2 HPO 4 , 0.3 g KH 2 PO 4 , 0.012 g MgSO 4 ·7H 2 O, and 0.1 g NH 4 Cl in 90 ml distilled water and 10 ml 0.1 g/l glucose solution sterilized by filtration), and finally cultivated for 24 h at 37°C while being stirred. Bacteria cultures in the lag phase and the early exponential phase of growth were used for inoculation of separated droplets in the micro fluidic system.…”

“…A second substance can have no effect on the first regarded substance, but it can also enhance or reduce their toxicity. In many cases, a change in the dose-response function is observed [3,4]. A reliable analysis of toxic effects in dependence of concentrations of two or more substances demands for a toxicological screening in a two-or higher-dimensional concentration space.…”

Microfluidics for cell-based high throughput screening platforms—A review

“…Cao et al [64] presented a microfluidic droplet device for the generation of multidimensional concentrations of antibiotics, to achieve an assay for toxic effects of Escherichia coli, see Fig. 12.…”

Droplet Microfluidic Technique for the Study of Fermentation

“…Droplet microfluidic systems offer powerful tools in screening of reaction conditions, e.g., Cao et al [14,15] and Churski et al [16] independently presented the use of such systems in screening of two-dimensional interactions between antibiotics. Earlier, Song et al [17], Damean et al [18], Niu et al [19], Sun et al [20], Wang et al [21] and Agresti et al [22] reported systems to generate sequences of droplets scanning concentrations of one substance (up to four orders of magnitude) and used them to screening of biochemical reactions.…”