Micro-Technologies for Assessing Microbial Dynamics in Controlled Environments.

With recent advances in microfabrication technologies, the miniaturization of traditional culturing techniques has provided ideal methods for interrogating microbial communities in a confined and finely controlled environment. Micro-technologies offer high-throughput screening and analysis, reduced experimental time and resources, and have low footprint. More importantly, they provide access to culturing microbes in situ in their natural environments and similarly, offer optical access to real-time dynamics under a microscope. Utilizing micro-technologies for the discovery, isolation and cultivation of "unculturable" species will propel many fields forward; drug discovery, point-of-care diagnostics, and fundamental studies in microbial community behaviors rely on the exploration of novel metabolic pathways. However, micro-technologies are still largely proof-of-concept, and scalability and commercialization of micro-technologies will require increased accessibility to expensive equipment and resources, as well as simpler designs for usability. Here, we discuss three different miniaturized culturing practices; including microarrays, micromachined devices, and microfluidics; advancements to the field, and perceived challenges.

Developing a Functional Poly(dimethylsiloxane)-Based Microbial Nanoculture System Using Dimethylallylamine.

Here, a novel poly(dimethylsiloxane) (PDMS)-based microbial culture system was investigated. Bacteria were encapsulated in functional and semipermeable membranes, mimicking the cell microenvironment and facilitating mass transport for interrogating microbial dynamics, thereby overcoming one of the major challenges associated with commercially available PDMS such as Sylgard 184. The hydrophobic nature and lack of control in the polymer network in Sylgard 184 significantly impede the the tunability of the transport and mechanical properties of the material as well as its usage as an isolation chamber for culturing and delivering microbes. Therefore, a novel PDMS composition was developed and functionalized with dimethylallylamine (DMAA) to alter its hydrophobicity and modify the polymer network. Characterization techniques including NMR spectroscopy, contact angle measurements, and sol-gel process were utilized to evaluate the physical and chemical properties of the newly fabricated membranes. Furthermore, the DMAA-containing polymer mixture was used as a proof of concept to generate hydrodynamically stable microcapsules and cultivate Escherichia coli cells in the functionalized capsules. The membrane exhibited a selective permeability to tetracycline, which diffused into the capsules to inhibit the growth of the encapsulated microbes. The functionality achieved here with the addition of DMAA, coupled with the high-throughput encapsulation technique, could prove to be an effective testing and diagnostic tool to evaluate microbial resistance, growth dynamics, and interspecies interaction and lays the foundation for in vivo models.

Employing LiCl salt gradient in the wild-type α-hemolysin nanopore to slow down DNA translocation and detect methylated cytosine.

In this research, we demonstrate a label-free detection, biological nanopore-based method to distinguish methylated cytosine (mC) from naked cytosine (C) in sample mixtures containing both C and mC at a prolonged translocation duration. Using a 15-fold increase in LiCl salt concentration going from a cis to trans chamber, we increased the translocation dwell time of ssDNA by over 5-fold and the event capture rate by 6-fold in comparison with the symmetric concentration of 1.0 M KCl (control). This is a consequence of counter-ion binding and effective lowering of the overall charge of DNA, which in turn lessens the electrophoretic drive of the system and slows the translocation velocity. Moreover, salt gradients can create a large electric field that will funnel ions and polymers towards the pore, increasing the capture rate and translocation dwell time of DNA. As a result, in 0.2 M-3.0 M LiCl solution, ssDNA achieved a prolonged dwell time of 52 µs per nucleotide and a capture rate of 60 ssDNA per second. Importantly, lowering the translocation speed of ssDNA enhances the resulting resolution, allowing 5'-mC to be distinguished from C without using methyl-specific labels. We successfully distinguished 5'-mC from C when mixed together at ratios of 1 : 1, 3 : 7 and 7 : 3. The distribution of current blockade amplitudes of all mixtures adopted bimodal shapes, with peak-to-peak ratios coarsely corresponding to the mixture composition (e.g. the density and distribution of events shifted in correspondence with changes in 18b-0mC and 18-2mC concentration ratios in the mixture).

Investigation of compacted DNA structures induced by Na+ and K+ monovalent cations using biological nanopores.

In aqueous solutions, an elongated, negatively charged DNA chain can quickly change its conformation into a compacted globule in the presence of positively charged molecules, or cations. This well-known process, called DNA compaction, is a method with great potential for gene therapy and delivery. Experimental conditions to induce these compacted DNA structures are often limited to the use of common compacting agents, such as cationic surfactants, polymers, and multivalent cations. In this study, we show that in highly concentrated buffers of 1 M monovalent cation solutions at pH 7.2 and 10, biological nanopores allow real-time sensing of individual compacted structures induced by K+ and Na+, the most abundant monovalent cations in human bodies. Herein, we studied the ratio between compacted and linear structures for 15-mer single-stranded DNA molecules containing only cytosine nucleotides, optimizing the probability of linear DNA chains being compacted. Since the binding affinity of each nucleotide to cation is different, the ability of the DNA strand to fold into a compacted structure greatly depends on the type of cations and nucleotides present. Our experimental results compare favorably with findings from previous molecular dynamics simulations for the DNA compacting potential of K+ and Na+ monovalent cations. We estimate that the majority of single-stranded DNA molecules in our experiment are compacted. From the current traces of nanopores, the ratio of compacted DNA to linear DNA molecules is approximately 30 : 1 and 15 : 1, at a pH of 7.2 and 10, respectively. Our comparative studies reveal that Na+ monovalent cations have a greater potential of compacting the 15C-ssDNA than K+ cations.