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An overview of the workflow of single-cell isolation, lysis, and DNA amplification in a microfluidic chip. The blue rectangles represent standard steps in an on-chip SC-WGA process. The green box highlights the general steps of the lysis protocol developed and optimized.
Source publication
Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mo...
Citations
... Integrating microfluidics into CRC diagnostics would enable the detection of low-abundance-specific biomarkers or genetic mutations associated with CRC, minimizing the likelihood of false-positive or false-negative results. Moreover, microfluidic platforms can precisely manipulate bacterial and human cells with single-cell resolution, offering the potential to shed new light on the intricate mechanisms underlying CRC development and progression through the analysis of the genome and transcriptome [13][14][15]. As a result, leveraging microfluidic technologies, it is the promise of future novel diagnostic approaches to include early detection, treatment response monitoring, and minimal residual disease detection in CRC patients to better ensure long-term remission. ...
Colorectal cancer (CRC) is a prevalent and potentially fatal disease categorized based on its high incidences and mortality rates, which raised the need for effective diagnostic strategies for the early detection and management of CRC. While there are several conventional cancer diagnostics available, they have certain limitations that hinder their effectiveness. Significant research efforts are currently being dedicated to elucidating novel methodologies that aim at comprehending the intricate molecular mechanism that underlies CRC. Recently, microfluidic diagnostics have emerged as a pivotal solution, offering non-invasive approaches to real-time monitoring of disease progression and treatment response. Microfluidic devices enable the integration of multiple sample preparation steps into a single platform, which speeds up processing and improves sensitivity. Such advancements in diagnostic technologies hold immense promise for revolutionizing the field of CRC diagnosis and enabling efficient detection and monitoring strategies. This article elucidates several of the latest developments in microfluidic technology for CRC diagnostics. In addition to the advancements in microfluidic technology for CRC diagnostics, the integration of artificial intelligence (AI) holds great promise for further enhancing diagnostic capabilities. Advancements in microfluidic systems and AI-driven approaches can revolutionize colorectal cancer diagnostics, offering accurate, efficient, and personalized strategies to improve patient outcomes and transform cancer management.
... All extraction kits yielded a higher abundance of Gram-negative bacteria than Gram-positive. Grampositive bacteria contain a thicker and stronger peptidoglycan layer making them harder to lyse (Liu et al., 2018), which results in the limited detection of Gram-positive bacteria within a microbial Frontiers in Microbiology 12 frontiersin.org community (Hermans et al., 2018). ...
Introduction
Fluctuations in environmental physicochemical parameters can affect the diversity and prevalence of microbial communities, including vibrios, associated with aquatic species and their surrounding environments. This study aimed to investigate the population dynamics of two Vibrio species as well as the microbial community diversity of whole crab and seawater from the Maryland Coastal Bays (MCBs), using 16S rRNA sequencing.
Methods
During this study, three crabs and 1 L of seawater were collected monthly from two sites for 3 months. Crab tissue was extracted and pooled for each site. Extracted crab tissue and seawater were analyzed for Vibrio parahaemolyticus and V. vulnificus using Most Probable Number (MPN) real-time PCR. For 16S rRNA microbiome analysis, three different DNA extraction kits were evaluated to extract microbial DNA from individual crabs. Also, 500 mL of each seawater sample was filtered for DNA extraction.
Results
Results indicated that sample types and sampling periods had a significant effect on the alpha diversity of the microbial community of crabs and seawater ( p < 0.05); however, no statistical difference was found between DNA extraction kits. Beta diversity analysis also found that the microbial compositions between sample types and temporal distributions were statistically significant. Taxonomic classification revealed that Proteobacteria, Cyanobacteria, Actinobacteria, and Bacteroidetes were present in both crab and seawater samples. Vibrio parahaemolyticus and V. vulnificus were also detected in both crab and seawater samples, although crabs contained a higher concentration of the bacterium compared to the seawater samples. It was found that vibrios were not a dominant species in the microbial community of crab or seawater samples.
Discussion
Results from this study provide further insight into species diversity and phylogenetic compositions of blue crabs and seawater from the MCBs. These approaches will help in risk assessments that are essential in the overall advancement of public health.
... The simultaneous manipulation of individual bacteria while obtaining expression data still faces many challenges [36,104]. On the other hand, the tough cell wall in Gram-positive bacteria hinders the isolation of their nucleic acids, which is also a common difficulty [105]. ...
The recurrence of bacterial infectious diseases is closely associated with bacterial persisters. This subpopulation of bacteria can escape antibiotic treatment by entering a metabolic status of low activity through various mechanisms, for example, biofilm, toxin-antitoxin modules, the stringent response, and the SOS response. Correspondingly, multiple new treatments are being developed. However, due to their spontaneous low abundance in populations and the lack of research on in vivo interactions between persisters and the host's immune system, microfluidics, high-throughput sequencing, and microscopy techniques are combined innovatively to explore the mechanisms of persister formation and maintenance at the single-cell level. Here, we outline the main mechanisms of persister formation, and describe the cutting-edge technology for further research. Despite the significant progress regarding study techniques, some challenges remain to be tackled.
... Microfluidics offers a promising solution for this problem [7][8][9][10][11] . Microfluidic systems can identify cells based on images 12,13 , fluorescence signals [14][15][16] , or Raman spectrum [17][18][19] , and further sort the target cells via DEP 20,21 , gravity 22 , centrifugal force 23 , optical tweezer 18,24,25 , or acoustic waves 26,27 . ...
Identification, sorting, and sequencing of individual cells directly from in situ samples have great potential for in-depth analysis of the structure and function of microbiomes. In this work, based on an artificial intelligence (AI)-assisted object detection model for cell phenotype screening and a cross-interface contact method for single-cell exporting, we developed an automatic and index-based system called EasySort AUTO, where individual microbial cells are sorted and then packaged in a micro-droplet and automatically exported in a precisely indexed, "One-Cell-One-Tube" manner. The target cell is automatically identified based on an AI-assisted object detection model and then mobilized via an optical tweezer for sorting. Then, a cross-interface contact microfluidic printing method that we developed enables the automated transfer of cells from the chip to the tube, which leads to coupling with subsequent single-cell culture or sequencing. The efficiency of the system for single-cell printing is >93%. The throughput of the system for single-cell printing is~120 cells/h. Moreover, >80% of single cells of both yeast and Escherichia coli are culturable, suggesting the superior preservation of cell viability during sorting. Finally, AI-assisted object detection supports automated sorting of target cells with high accuracy from mixed yeast samples, which was validated by downstream single-cell proliferation assays. The automation, index maintenance, and vitality preservation of EasySort AUTO suggest its excellent application potential for single-cell sorting.
... Representative bright field images of the sample before and after sorting of a bacterial cell are shown in Figure 2B. A notorious technical challenge in single bacterium sequencing is the rigid cell wall in Gram-positive bacteria, which makes the isolation of their genome very difficult 18 . To improve the cell lysis efficiency, we optimized several steps in the protocol, including the pretreatment step (freeze-thaw cycles), lysis buffer, and incubation time (see Materials and methods section for details). ...
Despite the fast progress in our understanding of the complex functions of gut microbiota, it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis. To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput, here, we propose an integrative strategy, which combines the use of fluorescent probe labeling, confocal imaging with single‐cell sorting, and sequencing. Mouse gut bacteria sequentially labeled by two fluorescent d‐amino acid probes in vivo were first imaged by confocal microscopy to visualize their growth patterns, which can be unveiled by the distribution of the two fluorescence signals on each bacterium. Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment, and the collected cells were then sequenced individually to identify their taxa. Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single‐cell basis, which should also have great potential in studying many other complex bacterial systems.
... coli) replicates giving amplification for 10 CFU. This reduced sensitivity is expected, as thermal lysis (95°C, 2 min), performed to disrupt the bacterial cell wall (Materials and Methods), has been previously shown to be inefficient in lysing bacteria (50,51). Next, we repeated the above experiments with pathogens in blood in the biphasic reaction format for MRSA (SI Appendix, Fig. S10E and Fig. 3F) and E. coli (SI Appendix, Fig. 3G). ...
Blood stream infections (BSIs) cause high mortality, and their rapid detection remains a significant diagnostic challenge. Timely and informed administration of antibiotics can significantly improve patient outcomes. However, blood culture, which takes up to 5 d for a negative result, followed by PCR remains the gold standard in diagnosing BSI. Here, we introduce a new approach to blood-based diagnostics where large blood volumes can be rapidly dried, resulting in inactivation of the inhibitory components in blood. Further thermal treatments then generate a physical microscale and nanoscale fluidic network inside the dried matrix to allow access to target nucleic acid. The amplification enzymes and primers initiate the reaction within the dried blood matrix through these networks, precluding any need for conventional nucleic acid purification. High heme background is confined to the solid phase, while amplicons are enriched in the clear supernatant (liquid phase), giving fluorescence change comparable to purified DNA reactions. We demonstrate single-molecule sensitivity using a loop-mediated isothermal amplification reaction in our platform and detect a broad spectrum of pathogens, including gram-positive methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria, gram-negative Escherichia coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection (LOD) of 1.2 colony-forming units (CFU)/mL from 0.8 to 1 mL of starting blood volume. We validated our assay using 63 clinical samples (100% sensitivity and specificity) and significantly reduced sample-to-result time from over 20 h to <2.5 h. The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.
... Representative bright field images of the sample before and after sorting of a bacterial cell are shown in Figure 2B. A notorious technical challenge in single bacterium sequencing is the rigid cell wall in Gram-positive bacteria, which makes the isolation of their genome very difficult 18 . To improve the cell lysis efficiency, we optimized several steps in the protocol, including the pretreatment step (freeze-thaw cycles), lysis buffer, and incubation time (see Materials and methods section for details). ...
Despite the fast progress in our understanding of the complex functions of gut microbiota, it is still challenging to directly investigate the in vivo microbial activities and processes on an individual cell basis. To gain knowledge of the indigenous growth/division patterns of the diverse mouse gut bacteria with a relatively high throughput, here, we propose an integrative strategy, which combines the use of fluorescent probe labeling, confocal imaging with single-cell sorting, and sequencing. Mouse gut bacteria sequentially labeled by two fluorescent D-amino acid probes in vivo were first imaged by confocal mi-croscopy to visualize their growth patterns, which can be unveiled by the distribution of the two fluorescence signals on each bacterium. Bacterial cells of interest on the imaging slide were then sorted using a laser ejection equipment, and the collected cells were then sequenced individually to identify their taxa. Our strategy allows integrated acquirement of the growth pattern knowledge of a variety of gut bacteria and their genomic information on a single-cell basis, which should also have great potential in studying many other complex bacterial systems. Keywords: bacterial growth pattern; confocal imaging; fluorescent D-amino acid probe; in vivo labeling; single-cell sequencing Impact statement Bacteria residing in the mammalian gut have diverse cellular morphologies, but our knowledge of this topic is still very limited. Previously, we developed an in vivo labeling protocol where fluorescent D-amino acid probes were sequentially given to mouse via gavage to record the in situ growing processes of its gut microbes. However, identifying the taxa of a particular bacterium that showed interesting growth or division modes was still very challenging. Here, we integrate the use of confocal fluorescence imaging with single-cell sorting and sequencing, which allows directly isolating the cell of interest under a confocal microscope and then identifying its taxa.
... Microfluidic experimental setup. SC-WGA was performed in an optofluidic platform at Mayo Clinic (Rochester, MN) [11][12][13] with a customized microfluidic device for high-throughput SC-WGA. Briefly, this platform consists of a microscope (Nikon Eclipse), optical tweezers (Thorlabs) and a custom-built Polydimethylsiloxane (PDMS) microfluidic device with 4 identical reaction blocks that contain 14 parallel reaction lines in each block (Fig. 2a). ...
... A representative image of using the laser tweezers to trap a single Nostoc cell is shown in Fig. 2c. Lysis of the Nostoc cells and genome amplification procedure followed our optimized protocol for bacterial single cell lysis and whole genome amplification in microfluidic devices for downstream sequencing 11 . The protocols for Nostoc lysis and library construction and sequencing can be found in SI. ...
Understanding the impact of long-term exposure of microorganisms to space is critical in understanding how these exposures impact the evolution and adaptation of microbial life under space conditions. In this work we subjected Nostoc sp. CCCryo 231-06, a cyanobacterium capable of living under many different ecological conditions, and also surviving in extreme ones, to a 23-month stay at the International Space Station (the Biology and Mars Experiment, BIOMEX, on the EXPOSE-R2 platform) and returned it to Earth for single-cell genome analysis. We used microfluidic technology and single cell sequencing to identify the changes that occurred in the whole genome of single Nostoc cells. The variant profile showed that biofilm and photosystem associated loci were the most altered, with an increased variant rate of synonymous base pair substitutions. The cause(s) of these non-random alterations and their implications to the evolutionary potential of single bacterial cells under long-term cosmic exposure warrants further investigation.
... This micropillar array induced DNA extraction and immobilization, allowing for whole genome amplification (WGA) to be performed in a continual fluid flow on the chip while maintaining the ability to be removed from the chip for sequencing. Both gram-negative and gram-positive bacteria lysis and genomic DNA extraction have been done in a single microfluidic system [21]. The lysis procedure incorporated thermal, enzymatic, and chemical lysis methods and was followed by on-chip WGA. ...
The development of elegant and numerous microfluidic manipulations has enabled significant advances in the processing of small volume samples and the detection of minute amounts of biomaterials. Effective isolation of single cells in a defined volume as well as manipulations of complex bioparticle or biomolecule mixtures allows for the utilization of information-rich detection methods including mass spectrometry, electron microscopy imaging, and amplification/sequencing. The art and science of translating biosamples from microfluidic platforms to highly advanced, information-rich detection system is the focus of this review, where we term the translation between the microfluidics elements to the external world "off-chipping." When presented with the challenge of presenting sub-nanoliter volumes of manipulated sample to a detection scheme, several delivery techniques have been developed for effective analysis. These techniques include spraying (electrospray, nano-electrospray, pneumatic), meniscus-defined volumes (droplets, plugs), constrained volumes (narrow channels, containers), and phase changes (deposition, freezing). Each technique has been proven effective in delivering highly defined samples from microfluidic systems to the detection elements. This review organizes and presents selective publications that illustrate the advancements of these delivery techniques with respect to the type of sample analyzed, while introducing each strategy and providing historical perspective. The publications highlighted in this review were chosen due to their significance and relevance in the development of their respective off-chip technique. This review highlights advancements of delivery methods off a microfluidic chip for additional information rich detection schemes.
... This work was performed in our optofluidic platform at Mayo Clinic (Rochester, MN) (Liu et al., 2018(Liu et al., , 2019b with a customized microfluidic device for high-throughput SC-WGA. Briefly, this platform consists of a microscope (Nikon Eclipse), optical tweezers (Thorlabs) and a custom-built Polydimethylsiloxane (PDMS) microfluidic device with 4 identical reactions blocks that contains 14 parallel reaction lines in each block (Figure 1A). ...
The Nostoc sp. strain CCCryo 231-06 is a cyanobacterial strain capable of surviving under extreme conditions and thus is of great interest for the astrobiology community. The knowledge of its complete genome sequence would serve as a guide for further studies. However, a major concern has been placed on the effects of contamination on the quality of sequencing data without a reference genome. Here we report the use of microfluidic technology combined with single cell sequencing and de novo assembly to minimize the contamination and recover the complete genome of the Nostoc strain CCCryo 231-06 with high quality. 100% of the whole genome was recovered with all contaminants removed and a strongly supported phylogenetic tree. The data reported can be useful for comparative genomics for phylogenetic and taxonomic studies. The method used in this work can be applied to studies that require high-quality assemblies of genomes of unknown microorganisms.
