Whole lifecycle observation of single-spore germinated Streptomyces using a nanogap-stabilized microfluidic chip.

Streptomyces is a model bacterium to study multicellular differentiation and the major reservoir for antibiotics discovery. However, the cellular-level lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools that can mimic their natural conditions. In this study, we developed a simple microfluidic chip for the cultivation and observation of the entire lifecycle of Streptomyces development from the single-cell perspective. The chip consists of channels for loading samples and supplying nutrients, microwell arrays for the seeding and growth of single spores, and air chambers beside the microwells that facilitate the development of aerial hyphae and spores. A unique feature of this chip is that each microwell is surrounded by a 1.5 µm nanogap connected to an air chamber, which provides a stabilized water-air interface. We used this chip to observe the lifecycle development of Streptomyces coelicolor and Streptomyces griseus germinated from single spores, which revealed differentiation of aerial hyphae with progeny spores at micron-scale water-air interfaces and air chambers. Finally, we demonstrated the applicability of this chip in phenotypic assays by showing that the microbial hormone A-Factor is involved in the regulatory pathways of aerial hyphae and spore formation. The microfluidic chip could become a robust tool for studying multicellular differentiation, single-spore heterogeneity, and secondary metabolism of single-spore germinated Streptomyces.

Direct antimicrobial susceptibility testing of bloodstream infection on SlipChip.

This paper describes an integrated microfluidic SlipChip device for rapid antimicrobial susceptibility testing (AST) of bloodstream pathogens in positive blood cultures. Unlike conventional AST methods, which rely on an overnight subculture of positive blood cultures to obtain isolated colonies, this device enables direct extraction and enrichment of the bacteria from positive blood cultures by dielectrophoresis. SlipChip technology enables parallel inoculation of the extracted bacteria into nanoliter-scale broth droplets to perform multiplexed ASTs simultaneously. The nanoliter confinement in the droplets increases the effective inoculation amount of the bacteria, shortens the diffusion distance of nutrient elements and gases, and allows faster growth and proliferation rates. Entropy-based image analysis used for the characterization of bacterial susceptibility patterns eliminates the requirement for single-cell morphological analysis and fluorescence labeling. As a proof-of-concept, the susceptibility patterns of Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538p, and a positive blood culture containing Escherichia coli against several broad-spectrum antibiotics were determined by the SlipChip device. The on-chip AST results were well matched with those respectively reported by the broth microdilution method and a BD Phoenix Automated Microbiology System. Reliable AST results can be reported to clinicians within 3-8 h using this simple device after positive blood culture, allowing earlier proper administration of antimicrobial therapy.

Assembled Step Emulsification Device for Multiplex Droplet Digital Polymerase Chain Reaction.

Digital PCR is a powerful method for absolute nucleic acid quantification with unprecedented accuracy and precision. To promote the wider use and application of digital PCR, several major challenges still exist, including reduction of cost, integration of the instrumental platform, and simplification of operations. This paper describes a reusable microfluidic device that generates nanoliter droplet arrays based on step emulsification for the on-chip multiplex digital PCR of eight samples simultaneously. The device contains two glass plates that can be quickly assembled with prefilled mineral oil. Droplets are simply generated through the arrays of step emulsification nozzles driven by a single pressure controller and are self-assembled into monolayer droplet arrays in U-shaped chambers. The use of mineral oil eliminates bubble generation; thus, no overpressure is required during thermocycling. Moreover, the device can be reused many times after disassembly and a brief cleaning procedure, which significantly reduces the cost of the device per dPCR assays. The device was able to detect template DNA at concentrations as low as 10 copies/µL with a dynamic range of approximately 4 logs. We applied this device in the quantitative assessment of HER2 copy number variation, which is important for targeted therapy and prognosis of breast cancer. The performance was validated by 16 clinical samples, obtaining similar results to commercial digital PCR. We envision that this low-cost, reusable, and user-friendly device can be broadly used in various applications.

A microfluidic surface-enhanced Raman spectroscopy approach for assessing the particle number effect of AgNPs on cytotoxicity.

Silver nanoparticles (Ag NPs) have well-known antibacterial properties and are widely applied in various medical products and general commodities. Although many studies have addressed the toxicity of Ag NPs to mammalian cells, the direct relationship between the number of Ag NPs in living cells and the corresponding cell toxicity has not yet been explicitly demonstrated. In this work, a simple and reusable microfluidic device composed of a quartz cover slip and a glass plate with etched micro-channel and micro-wells was employed for separating and trapping single living cells. The device was silanized to render the surface hydrophobic. For simplicity, HeLa cells as the model cancer cells were used in the study, which were pipette-loaded into an array of micro wells based on dead-end filling. Surface enhanced Raman spectroscopy (SERS) was then employed to examine the living cancer cells and assessed number and distribution of Ag NPs in the cells. Combined with the cell viability assay, we therefore correlated the number of Ag NPs in the cell with the toxicity to the cell directly.

Dynamic Sessile-Droplet Habitats for Controllable Cultivation of Bacterial Biofilm.

Bacterial biofilms play essential roles in biogeochemical cycling, degradation of environmental pollutants, infection diseases, and maintenance of host health. The lack of quantitative methods for growing and characterizing biofilms remains a major challenge in understanding biofilm development. In this study, a dynamic sessile-droplet habitat is introduced, a simple method which cultivates biofilms on micropatterns with diameters of tens to hundreds of micrometers in a microfluidic channel. Nanoliter plugs are utilized, spaced by immiscible carrier oil to initiate and support the growth of an array of biofilms, anchored on and spatially confined to the micropatterns arranged on the bottom surface of the microchannel, while planktonic or dispersal cells are flushed away by shear force of aqueous plugs. The performance of the aforementioned method of cultivating biofilms is demonstrated by Pseudomonas aeruginosa PAO1 and its derived mutants, and quantitative antimicrobial susceptibility testing of PAO1 biofilms. This method could significantly eliminate corner effects, avoid microchannel clogging, and constrain the growth of biofilms for long-term observations. The controllable sessile droplet-based biofilm cultivation presented in this study should shed light on more quantitative and long-term studies of biofilms, and open new avenues for investigation of biofilm attachment, growth, expansion, and eradication.