Enrichment of different taxa of the enigmatic candidate phyla radiation bacteria using a novel picolitre droplet technique.

The candidate phyla radiation (CPR) represents a distinct monophyletic clade and constitutes a major portion of the tree of life. Extensive efforts have focused on deciphering the functional diversity of its members, primarily using sequencing-based techniques. However, cultivation success remains scarce, presenting a significant challenge, particularly in CPR-dominated groundwater microbiomes characterized by low biomass. Here, we employ an advanced high-throughput droplet microfluidics technique to enrich CPR taxa from groundwater. Utilizing a low-volume filtration approach, we successfully harvested a microbiome resembling the original groundwater microbial community. We assessed CPR enrichment in droplet and aqueous bulk cultivation for 30 days using a novel CPR-specific primer to rapidly track the CPR fraction through the cultivation attempts. The combination of soil extract and microbial-derived necromass provided the most supportive conditions for CPR enrichment. Employing these supplemented conditions, droplet cultivation proved superior to bulk cultivation, resulting in up to a 13-fold CPR enrichment compared to a 1- to 2-fold increase in bulk cultivation. Amplicon sequencing revealed 10 significantly enriched CPR orders. The highest enrichment in CPRs was observed for some unknown members of the Parcubacteria order, Cand. Jorgensenbacteria, and unclassified UBA9983. Furthermore, we identified co-enriched putative host taxa, which may guide more targeted CPR isolation approaches in subsequent investigations.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

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.

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.

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.

A droplet-based microfluidic immunosensor for high efficiency melamine analysis.

We report a droplet-based microfluidic immunosensor for the rapid and accurate detection of melamine, an organic base that has been implicated in widescale adulteration of food products such as milk. Our melamine assay is based on the competitive reaction between native melamine and a melamine-fluorescein isothiocyanate (FITC) conjugate against an anti-hapten antibody. The adoption of fluorescence polarization, allows the quantification of melamine in a more direct and rapid manner than established heterogeneous methods based on liquid chromatography, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). The detection protocol provides a limit of detection of 300 ppb, which is below the maximum allowable melamine levels (2.5 ppm) defined by the U.S. Food and Drug Administration and the European Commission to a significant extent.