Droplet microfluidics has several key features in microbiology research:
1. Encapsulation of microorganisms in droplets provides isolation, eliminating differences in growth rates and interspecific competition, facilitating the study of rare or slow-growing microorganisms, while rapid accumulation of metabolites in droplets activates concentration-dependent physiological pathways such as colony sensing
2. The microfluidic device generates highly homogeneous droplets at frequencies up to 20,000 Hz, enabling high-throughput analysis and thus making possible ultra-high-throughput identification and screening of microorganisms.
3. In the droplet microfluidic system, a variety of control modules can be designed and integrated into customized channels according to research needs to precisely manipulate droplet injection, mixing, dispersion, prolonged incubation, and sorting, and to rapidly introduce a variety of detection reagents and stimuli to create a diverse and controllable environment for high-throughput and precise control of microbial cells.
Droplet microfluidics for microorganisms consists of several main components:
1. Droplet generation, including the generation of single-phase dispersed droplets, droplet arrays and droplet surfaces or intra-droplet components made of different materials;
2. Manipulation of droplets and components within droplets;
3. Analysis of microorganisms within the droplet.
1. High-throughput single-cell culture and isolation of microorganisms
Conventional microbial culture methods are not only time-consuming and complicated, but also ignore some microbial information due to bacterial interspecies inhibition and slow or no growth of certain microorganisms.
Droplet microfluidics-based culture technology places a small group or even a single microorganism in a droplet, thereby avoiding interspecies competition, precisely controlling the microbial growth environment, and isolating microorganisms that cannot be cultured by conventional methods.
The isolation of rare or difficult-to-cultivate microorganisms from complex microbial communities is made easier by utilizing droplet microfluidics.
Microfluidic Plate Scribing (MSP) enables high-throughput isolation and culture of single microorganisms, where droplets are scribed on petri dishes pre-filled with carrier oil to form stable droplet arrays.
Microfluidic plate scribing offers several advantages over traditional agar plate scribing:
1. Higher culture throughput and lower reagent and sample consumption;
2. Eliminate the effects of interspecific competition and differences in growth rates;
3. Droplets on carrier oil can support up to 5 months of single-cell culture;
4. Allow microscopic observation during incubation;
5. Cultivation can be followed by next-generation sequencing for microbial community analysis and expanded culture;
6. MSP uses a liquid culture medium to avoid hydrogen peroxide produced during agar solidification, which facilitates the isolation and culture of hydrogen peroxide-sensitive microorganisms.
2. High-throughput detection, characterization and microbiome studies of microorganisms
1) Optical droplet detection technology
Combining fluorescence in situ hybridization (FISH) with droplet microfluidics enables high-throughput microbial detection and identification.
To avoid the potential impact of fluorescent labeling on the normal physiological activities of bacteria, the label-free and noninvasive single-cell Raman spectroscopy (SCRS) technique was used for microbial detection in microdroplets.
SCRS is a metabolite detection technology based on molecular vibrational signaling that provides an endogenous biochemical “fingerprint” of individual cells.
By combining SCRS with genome sequencing technology, the genome and metabolome of individual cells can be systematically studied, providing a powerful tool for in-depth analysis of microorganisms.
2) Droplet Digital PCR
The introduction of droplet microfluidics has dramatically revolutionized digital PCR technology, giving rise to droplet digital PCR (ddPCR).
In ddPCR, DNA molecules are randomly dispensed into each droplet for routine PCR. since the reaction mixture contains fluorescent probes, the copy number of the target DNA in the original sample can be calculated from the fluorescence intensity, enabling the detection and quantification of the target microorganisms in the sample.
ddPCR is capable of determining the absolute abundance of individual microbial taxa in complex communities, diagnosing early viral infections, as well as quantitatively detecting BK virus, Hepatitis B virus, and human papillomavirus.
3) Droplet Sequencing for Microbiome Research
Combining droplet microfluidics with next-generation sequencing technology avoids the heterogeneity at the single-cell level and the loss of spatial microbiome information that characterizes traditional 16S rRNA sequencing and macrogenome sequencing.
This combination has given rise to the MaPS-seq technique, which offers the prospect of studying the spatial distribution of microorganisms in complex environments. The technique has been applied to microbiome studies in different regions of the mouse gut, revealing multiple distributional relationships among specific populations.
Subsequently, Microbe-seq, a high-throughput single-cell genomics technology for microorganisms based on droplet microfluidics, was developed, which is capable of acquiring genomic information from a large number of single microbial cells and pinpointing the genomic information of microbial communities down to the strain level.
3. Droplet-based co-culture techniques for studying microbial interactions
Microbial interactions are key drivers of microbial community function and dynamics. Microbial co-culture using droplet microfluidics is well suited for high-throughput studies of microbial interactions in different environments.
This technique can also be used to study cross-border interactions of microorganisms. Droplet-based high-throughput co-culture and isolation techniques can effectively screen probiotics with antimicrobial activity.
4. Antibiotic susceptibility testing
Microfluidic-based bacterial culture methods are promising tools for performing automated and rapid antibiotic susceptibility testing.
In recent years, various microfluidic techniques such as microchambers, microchannels, and droplet-based methods have been developed for antibiotic susceptibility testing, among which droplet-based methods have been widely used due to their ability to generate large-scale, ultra-small-volume droplets, which are suitable for both high-throughput and high-sensitivity testing.
In addition, droplet microfluidics can be used to evaluate antiviral drugs. For example, to determine the effect of latency reversal agents on HIV reactivation, researchers developed a microfluidic device for high-throughput encapsulation of HIV-infected cells and direct characterization of these cells.
By analyzing individual CD4+ T cells from patients receiving antiretroviral therapy, the study found that latency reversal agents enhanced transcription of activated cells in some cases and increased the number of transcriptionally active cells in others.
5. Engineering strains and microbial resource development
Droplet microfluidics provides an efficient platform for strain improvement, metabolite screening and discovery of biosynthetic gene clusters. Combining macrogenomic library construction with droplet screening helps to discover biosynthetic gene clusters from microorganisms.
In addition, droplet microfluidics can be used for directed evolution, a common approach in protein engineering that directs proteins or nucleic acids to achieve a desired function by mimicking the process of natural selection.
The advantages of droplet microfluidics for high-throughput sorting make it ideal for performing directed evolution.
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