Jialan Cao

Cultivation of Chlorella vulgaris in microfluid segments and microtoxicological determination of their sensitivity against CuCl2 in the nanoliter range

The cultivation of the monocellular green alga Chlorella vulgaris was implemented into microfluid segments to demonstrate the possibility of an automated screening of toxic effects of the common algaecide CuCl2. Therefore, the nutritional as well as light and carbon dioxide requirements of the algae had to be adapted to the microfluidic device. Generally, sequences of about 350 fluid segments with single volumes of about 500 nL were applied for the dose–response experiments. The growth of algae cultures inside microfluidic segments was non‐invasively measured by microflow through techniques using two different optical channels. A multi‐endpoint detection was realized by the photometric characterization of cell density by transmission measurements and the measurement of density of autofluorescent cells. The different methods revealed comparable half maximal effective concentrations (EC50) in the range between 34.6 and 39.9 μg/mL for the toxicity of CuCl2 to the green algae C. vulgaris. By reference experiments in microtiter plates lower EC50 were achieved presumably caused by increased alkalinity of the growth medium due to higher photosynthesis. The results show that the microsegmented flow technique is well suited for the automated determination of dose/response functions for microorganisms like C. vulgaris and for the application of multi‐endpoint procedures at the nanoliter scale.

Droplet‐based microfluidics for microtoxicological studies

The specific sensitivity of different organisms, cells, and tissues against chemicals, the huge number of produced and applied substances, and the combinatorial explosion of effects by the combination of substances demand new concepts for the efficient determination of toxicological dose/response relations. The generation, processing, and characterization of microdroplets of different chemical compositions enable microfluidic approaches to offer a very promising strategy for strongly miniaturized toxicological screenings. Over the last decade, several developments demonstrated the applicability of droplet‐based microfluidic systems for toxicological studies. We review the current state of developments in microfluidics for toxicological applications. These have become powerful alternatives to microtiter plates and are very promising for replacing the standard methods in many fields. This article will focus on toxicological dose/response screenings with the variation of droplet composition, particularly on the possibility of generating “concentration spaces,” as well as on the applied sensing principles of organisms and cells inside the microdroplets.

Application of micro-segmented flow for two-dimensional characterization of the combinatorial effect of zinc and copper ions on metal-tolerant Streptomyces strains.

The cultivation and growth behavior of metal-tolerant strains of Streptomyce acidiscabies E13 and Streptomyces sp. F4 were studied under droplet-based microfluidics conditions. It was shown that the technique of micro segmented flow is well suited for the investigation of dependence of bacterial growth on different concentrations of either single metal ions or combinations of them. This study confirms higher tolerance to Zn than to Cu by our test organism. The highly resolved dose–response curves reflect two transitions between the different growth behaviors, separating initial responses to Cu concentration ranges into those with (a) intense growth, (b) moderate growth, and (c) growth inhibition. For Streptomyces sp. F4, an initial stimulation was shown in the sublethal range of zinc sulfate. Two-dimensional screenings using computer-controlled fluid actuation and in situ micro flow-through fluorimetry reflected a strong growth stimulation of strain F4 by zinc sulfate in the presence of sublethal Cu concentrations. This stimulatory effect on binary mixtures may be useful in providing optimal growth conditions in bioremediation procedures.

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

Abstract Droplet-based microfluidic technique is very suitable for a variety of screening processes. The cultivation within nanoliter segments and multidimensional microtoxicological screening of the Gram-negative bacterium Escherichia coli were studied under droplet-based microfluidic conditions. In order to evaluate the toxicity of the binary and ternary mixtures of antibiotic ampicillin and caffeine in the presence of the angiotensin-converting enzyme inhibitor captopril, a time-resolved optical double endpoint detection unit was applied. It included a microflow-through fluorimeter and photometer, which can simultaneously analyze changes in the endogenous autofluorescence signal and the cell density of E. coli cultivated inside 450-nl microfluid segments. As a result, strong nonlinear combination effects and a concentration-dependent antagonistic effect, as well as formation of activity summits on bolographic maps, were found. Our findings confirm the importance of multiparameter investigations for toxicological studies and could be taken into account in medical practice.

Changing growth behavior of heavy‐metal tolerant bacteria: Media optimization using droplet‐based microfluidics

Overproduction of secondary metabolites requires know‐how for media optimization and design of effective screening procedures. Micro‐segmented flow was applied to test the growth characteristics of heavy‐metal tolerant soil bacteria (Bacillus sporothermodurans and Streptomyces tendae) toward changing media composition. The applied microfluidic technique was shown to be suitable for media optimization, with effects of media solutes like phosphorus and nitrogen, and a trace metal solution. For inorganic phosphate and trace metals, the synthesis of secondary metabolites had a much narrower tolerance for concentration ranges than vegetative cells. As we worked with metal‐tolerant strains, some tests were done in the presence of copper‐II ions in the bacteria‐specific critical concentration of 0.25 mM. In particular, for the Bacillus strain, the macronutrient phosphorus resulted in a threefold increase in growth with rising concentration. Copper led to a prolonged lag phase. For S. tendae, no similarly enhancing effect was observed. The trace metal solution promoted bacterial growth only in the case of S. tendae, for higher concentration ranges. For the nitrogen source, especially S. tendae responded with changes between the light scattering and the fluorescence signal. This could be a hint for the production of fluorescent secondary metabolites.

Stochastically reduced communities-Microfluidic compartments as model and investigation tool for soil microorganism growth in structured spaces

Microbial community in soil is a complex and dynamic system. Using traditional culture experiments it is difficult to model the stochastic distribution of single organisms of microbial communities in the soil pores structure. Droplet‐based micro‐segmented flow technique allows the transfer of the principle of stochastic confinement of stochastically reduced communities from soil micro pores into nanoliter droplets. Microfluidics was applied for the investigation and comparison of soil samples from ancient mining areas by highly resolved concentration‐dependent screenings. As results, the generation, incubation, and in situ optical characterization of nanoliter droplets of suspensions of unknown soil microbial communities allowed the identification of different response characteristics toward heavy metal exposition. The investigations proved the high potential of microfluidics for investigations of soil microbial communities. It may be in the future helpful to detect bacteria and consortia with special biosorption characteristics, which could be useful for the development of biological accumulation and detoxification strategies.

Mikrofluidik – großes Potenzial in kleinen Tropfen

The technique of micro segmented flow is well applicable for the investigation of soil microorganisms. It supplies highly resolved dose/ response functions for whole microbial communities as well as for single strains. In addition, it can be used in the search for microbial strains with special tolerance features, for example for enhanced concentrations of cobalt and nickel. Such strains have been found in soil samples from the surface of ancient copper mining areas and metal-contaminated soils from archaeological excavations.

Addressing of Concentration Spaces for Bioscreenings by Micro Segmented Flow with Microphotometric and Microfluorimetric Detection

Microfluid segments allow the efficient realization and application of well-separated test volumes for high-resolved and multidimensional investigations. With typical volumes in the nanoliter and lower microliter range, screening runs with several hundred up to several thousand single volumes can be realized with a total consumption of less than 1 mL test solution. The fluid segments act as confinements for the determination of the dose-related cell response on different effectors which can be applied in a precision better than 1% in concentration. One, two, or higher dimensional concentration spaces are addressed by PC-controlled low-pulsation syringe pumps. Micro flow-through photometric measurements allow the characterization of the quality of segment sequences and the determination of up to four optical channels with typical measurement frequencies between 500 and 5,000 Hz. The generation and characterization of microfluid segment sequences for screening purposes, the realization of different concentration spaces for the determination of effects of single substances and combinatorial effects, and the cultivation of different organisms are reported. The investigations have shown the applicability of micro segmented flow for fast microtoxicological screenings with prokaryotic and eukaryotic microorganisms like E. Coli, Chlorella vulgaris, and Saccharomyces cerevisiae and for multicellular systems like embryos of Danio rerio.