Advantages of optical fibers for facile and enhanced detection in droplet microfluidics.

Droplet microfluidics offers a unique opportunity for ultrahigh-throughput experimentation with minimal sample consumption and thus has obtained increasing attention, particularly for biological applications. Detection and measurements of analytes or biomarkers in tiny droplets are essential for proper analysis of biological and chemical assays like single-cell studies, cytometry, nucleic acid detection, protein quantification, environmental monitoring, drug discovery, and point-of-care diagnostics. Current detection setups widely use microscopes as a central device and other free-space optical components. However, microscopic setups are bulky, complicated, not flexible, and expensive. Furthermore, they require precise optical alignments, specialized optical and technical knowledge, and cumbersome maintenance. The establishment of efficient, simple, and cheap detection methods is one of the bottlenecks for adopting microfluidic strategies for diverse bioanalytical applications and widespread laboratory use. Together with great advances in optofluidic components, the integration of optical fibers as a light guiding medium into microfluidic chips has recently revolutionized analytical possibilities. Optical fibers embedded in a microfluidic platform provide a simpler, more flexible, lower-cost, and sensitive setup for the detection of several parameters from biological and chemical samples and enable widespread, hands-on application much beyond thriving point-of-care developments. In this review, we examine recent developments in droplet microfluidic systems using optical fiber as a light guiding medium, primarily focusing on different optical detection methods such as fluorescence, absorbance, light scattering, and Raman scattering and the potential applications in biochemistry and biotechnology that are and will be arising from this.

Droplet Microfluidics for Microbial Biotechnology.

Droplet microfluidics has recently evolved as a prominent platform for high-throughput experimentation for various research fields including microbiology. Key features of droplet microfluidics, like compartmentalization, miniaturization, and parallelization, have enabled many possibilities for microbiology including cultivation of microorganisms at a single-cell level, study of microbial interactions in a community, detection and analysis of microbial products, and screening of extensive microbial libraries with ultrahigh-throughput and minimal reagent consumptions. In this book chapter, we present several aspects and applications of droplet microfluidics for its implementation in various fields of microbial biotechnology. Recent advances in the cultivation of microorganisms in droplets including methods for isolation and domestication of rare microbes are reviewed. Similarly, a comparison of different detection and analysis techniques for microbial activities is summarized. Finally, several microbial applications are discussed with a focus on exploring new antimicrobials and high-throughput enzyme activity screening. We aim to highlight the advantages, limitations, and current developments in droplet microfluidics for microbial biotechnology while envisioning its enormous potential applications in the future.

Monitoring and external control of pH in microfluidic droplets during microbial culturing.

BACKGROUND: Cell-based experimentation in microfluidic droplets is becoming increasingly popular among biotechnologists and microbiologists, since inherent characteristics of droplets allow high throughput at low cost and space investment. The range of applications for droplet assays is expanding from single cell analysis toward complex cell-cell incubation and interaction studies. As a result of cellular metabolism in these setups, relevant physicochemical alterations frequently occur before functional assays are conducted. However, to use droplets as truly miniaturized bioreactors, parameters like pH and oxygen availability should be controlled similar to large-scale fermentation to ensure reliable research. RESULTS: Here, we introduce a comprehensive strategy to monitor and control pH for large droplet populations during long-term incubation. We show the correlation of fluorescence intensity of 6-carboxyfluorescein and pH in single droplets and entire droplet populations. By taking advantage of inter-droplet transport of pH-mediating molecules, the average pH value of several million droplets is simultaneously adjusted in an a priori defined direction. To demonstrate the need of pH control in practice, we compared the fermentation profiles of two E. coli strains, a K12-strain and a B-strain, in unbuffered medium with 5 g/L glucose for standard 1 L bioreactors and 180 pL droplets. In both fermentation formats, the commonly used B-strain E. coli BL21 is able to consume glucose until depletion and prevent a pH drop, while the growth of the K12-strain E. coli MG1655 is soon inhibited by a low pH caused by its own high acetate production. By regulating the pH during fermentation in droplets with our suggested strategy, we were able to prevent the growth arrest of E. coli MG1655 and obtained an equally high biomass yield as with E. coli BL21. CONCLUSION: We demonstrated a comparable success of pH monitoring and regulation for fermentations in 1 L scale and 180 pL scale for two E. coli strains. This strategy has the potential to improve cell-based experiments for various microbial systems in microfluidic droplets and opens the possibility for new functional assay designs.

One Sensor for Multiple Colors: Fluorescence Analysis of Microdroplets in Microbiological Screenings by Frequency-Division Multiplexing.

High-speed multiwavelength fluorescence measurements are of paramount importance in microfluidic analytics. However, multicolor detection requires an intricate arrangement of multiple detectors and meticulously aligned filters and dichroic beamsplitters that counteract the simplicity, versatility, and low cost of microfluidic approaches. To break free from the restrictions of optical setup complexity, we introduce a simpler single-sensor setup based on laser-frequency modulation and frequency-division multiplexing (FDM). We modulate lasers to excite the sample with four non-overlapping frequency signals. A single photomultiplier tube detects all the modulated emitted light collected by an optical fiber in the microfluidic chip. Signal demodulation is performed with a lock-in amplifier separating the emitted light into four color channels in real time. This approach not only reduces complexity and provides setup flexibility but also results in improved signal quality and, thus, higher signal-to-noise ratios that translate into increased sensitivity. To validate the setup for high-throughput biological applications, we measured multiple signals from different microorganisms and fluorescently encoded droplet populations for exploring beneficial or antagonistic roles in microbial cocultivation systems, as is the case for antibiotic screening assays.

Coding of Experimental Conditions in Microfluidic Droplet Assays Using Colored Beads and Machine Learning Supported Image Analysis.

To efficiently exploit the potential of several millions of droplets that can be considered as individual bioreactors in microfluidic experiments, methods to encode different experimental conditions in droplets are needed. The approach presented here is based on coencapsulation of colored polystyrene beads with biological samples. The decoding of the droplets, as well as content quantification, are performed by automated analysis of triggered images of individual droplets in-flow using bright-field microscopy. The decoding strategy combines bead classification using a random forest classifier and Bayesian inference to identify different codes and thus experimental conditions. Antibiotic susceptibility testing of nine different antibiotics and the determination of the minimal inhibitory concentration of a specific antibiotic against a laboratory strain of Escherichia coli are presented as a proof-of-principle. It is demonstrated that this method allows successful encoding and decoding of 20 different experimental conditions within a large droplet population of more than 105 droplets per condition. The decoding strategy correctly assigns 99.6% of droplets to the correct condition and a method for the determination of minimal inhibitory concentration using droplet microfluidics is established. The current encoding and decoding pipeline can readily be extended to more codes by adding more bead colors or color combinations.

Publisher Correction: Detection of antibiotics synthetized in microfluidic picolitre-droplets by various actinobacteria.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Detection of antibiotics synthetized in microfluidic picolitre-droplets by various actinobacteria.

The natural bacterial diversity is regarded as a treasure trove for natural products. However, accessing complex cell mixtures derived from environmental samples in standardized high-throughput screenings is challenging. Here, we present a droplet-based microfluidic platform for ultrahigh-throughput screenings able to directly harness the diversity of entire microbial communities. This platform combines extensive cultivation protocols in aqueous droplets starting from single cells or spores with modular detection methods for produced antimicrobial compounds. After long-term incubation for bacterial cell propagation and metabolite production, we implemented a setup for mass spectrometric analysis relying on direct electrospray ionization and injection of single droplets. Even in the presence of dense biomass we show robust detection of streptomycin on the single droplet level. Furthermore, we developed an ultrahigh-throughput screening based on a functional whole-cell assay by picoinjecting reporter cells into droplets. Depending on the survival of reporter cells, droplets were selected for the isolation of producing bacteria, which we demonstrated for a microbial soil community. The established ultrahigh-throughput screening for producers of antibiotics in miniaturized bioreactors in which diverse cell mixtures can be screened on the single cell level is a promising approach to find novel antimicrobial scaffolds.

3D-glass molds for facile production of complex droplet microfluidic chips.

In order to leverage the immense potential of droplet microfluidics, it is necessary to simplify the process of chip design and fabrication. While polydimethylsiloxane (PDMS) replica molding has greatly revolutionized the chip-production process, its dependence on 2D-limited photolithography has restricted the design possibilities, as well as further dissemination of microfluidics to non-specialized labs. To break free from these restrictions while keeping fabrication straighforward, we introduce an approach to produce complex multi-height (3D) droplet microfluidic glass molds and subsequent chip production by PDMS replica molding. The glass molds are fabricated with sub-micrometric resolution using femtosecond laser machining technology, which allows directly realizing designs with multiple levels or even continuously changing heights. The presented technique significantly expands the experimental capabilities of the droplet microfluidic chip. It allows direct fabrication of multilevel structures such as droplet traps for prolonged observation and optical fiber integration for fluorescence detection. Furthermore, the fabrication of novel structures based on sloped channels (ramps) enables improved droplet reinjection and picoinjection or even a multi-parallelized drop generator based on gradients of confinement. The fabrication of these and other 3D-features is currently only available at such resolution by the presented strategy. Together with the simplicity of PDMS replica molding, this provides an accessible solution for both specialized and non-specialized labs to customize microfluidic experimentation and expand their possibilities.

Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets.

The majority of today's antimicrobial therapeutics is derived from secondary metabolites produced by Actinobacteria. While it is generally assumed that less than 1% of Actinobacteria species from soil habitats have been cultivated so far, classic screening approaches fail to supply new substances, often due to limited throughput and frequent rediscovery of already known strains. To overcome these restrictions, we implement high-throughput cultivation of soil-derived Actinobacteria in microfluidic pL-droplets by generating more than 600,000 pure cultures per hour from a spore suspension that can subsequently be incubated for days to weeks. Moreover, we introduce triggered imaging with real-time image-based droplet classification as a novel universal method for pL-droplet sorting. Growth-dependent droplet sorting at frequencies above 100 Hz is performed for label-free enrichment and extraction of microcultures. The combination of both cultivation of Actinobacteria in pL-droplets and real-time detection of growing Actinobacteria has great potential in screening for yet unknown species as well as their undiscovered natural products.

Displacement-weighted velocity analysis of gliding assays reveals that Chlamydomonas axonemal dynein preferentially moves conspecific microtubules.

In vitro gliding assays, in which microtubules are observed to glide over surfaces coated with motor proteins, are important tools for studying the biophysics of motility. Gliding assays with axonemal dyneins have the unusual feature that the microtubules exhibit large variations in gliding speed despite measures taken to eliminate unsteadiness. Because axonemal dynein gliding assays are usually done using heterologous proteins, i.e., dynein and tubulin from different organisms, we asked whether the source of tubulin could underlie the unsteadiness. By comparing gliding assays with microtubules polymerized from Chlamydomonas axonemal tubulin with those from porcine brain tubulin, we found that the unsteadiness is present despite matching the source of tubulin to the source of dynein. We developed a novel, to our knowledge, displacement-weighted velocity analysis to quantify both the velocity and the unsteadiness of gliding assays systematically and without introducing bias toward low motility. We found that the quantified unsteadiness is independent of tubulin source. In addition, we found that the short Chlamydomonas microtubules translocate significantly faster than their porcine counterparts. By modeling the effect of length on velocity, we propose that the observed effect may be due to a higher rate of binding of Chlamydomonas axonemal dynein to Chlamydomonas microtubules than to porcine microtubules.