We studied the unzipping of single molecules of double-stranded DNA by pulling one of their two strands through a narrow protein pore. Polymerase chain reaction analysis yielded the first direct proof of DNA unzipping in such a system. The time to unzip each molecule was inferred from the ionic current signature of DNA traversal. The distribution of times to unzip under various experimental conditions fit a simple kinetic model. Using this model, we estimated the enthalpy barriers to unzipping and the effective charge of a nucleotide in the pore, which was considerably smaller than previously assumed.

Due to its relatively low level of antigenicity and high durability, titanium has successfully been used as the major material for biological implants. However, because the typical interface between titanium and tissue precludes adequate transmission of load into the surrounding bone, over time, load-bearing implants tend to loosen and revision surgeries are required. Osseointegration of titanium implants requires presentation of both biological and mechanical cues that promote attachment of and trigger mineral deposition by osteoblasts. While many factors contribute to differentiation, the relative importance of the various cues is unclear. To substantially improve osseointegration of titanium implants, we generated a gelatin methacryloyl (GelMA) scaffold, using an extrusion-based 3D bioprinter, which can be directly printed on and grafted to the titanium implant surface. We demonstrate that this scaffold is able to trigger mineral deposition of both MG63 osteoblasts and primary normal human osteoblasts in the absence of any exogenous osteogenic factors. Films of the same formulation failed to promote mineral deposition suggesting that the three dimensional scaffold was able to tip the balance in favor of differentiation despite other potentially unfavorable differentiation cues of the material. We further show that these GelMA lattices can be directly grafted to titanium alloy and are secure in vitro over a period of seven weeks. When grafted within a groove system, the GelMA hydrogel is protected from shearing forces in a marrow implantation model. This prepares the way for osteogenic coatings to be directly manufactured on the implant surface and packaged for surgery.

In this paper, we present a fully integrated lab-on-a-chip and associated instrument for the detection of bacteria from liquid samples. The system conducts bacterial lysis, nucleic acid isolation and concentration, polymerase chain reaction (PCR), and end-point fluorescent detection. To enable truly low-cost manufacture of the single-use disposable chip, we designed the plastic chip in a planar format without any active components to be amenable to injection molding and utilized a novel porous polymer monolith (PPM) embedded with silica that has been shown to lyse bacteria and isolate the nucleic acids from clinical samples (M. D. Kulinski, M. Mahalanabis, S. Gillers, J. Y. Zhang, S. Singh and C. M. Klapperich, Biomed. Microdevices, 2009, 11, 671–678).¹ The chip is made of Zeonex®, a thermoplastic with a high melting temperature to allow PCR, good UV transmissibility for UV-curing of the PPM, and low auto-fluorescence for fluorescence detection of the amplicon. We have built a prototype instrument to automate control of the fluids, temperature cycling, and optical detection with the capability of accommodating various chip designs. To enable fluid control without including valves or pumps on the chip, we utilized a remote valve switching technique. To allow fluid flow rate changes on the valveless chip, we incorporated speed changing fluid reservoirs. The PCR thermal cycling was achieved with a ceramic heater and air cooling, while end-point fluorescence detection was accomplished with an optical spectrometer; all integrated in the instrument. The chip seamlessly and automatically is mated to the instrument through an interface block that presses against the chip. The interface block aligns and ensures good contact of the chip to the temperature controlled region and the optics. The integrated functionality of the chip was demonstrated using Bacillus subtilis as a model bacterial target. A Taqman assay was employed on-chip to detect the isolated bacterial DNA.

The emergence and spread of bacterial resistance to ever increasing classes of antibiotics intensifies the need for fast phenotype-based clinical tests for determining antibiotic susceptibility. Standard susceptibility testing relies on the passive observation of bacterial growth inhibition in the presence of antibiotics. In this paper, we present a novel microfluidic platform for antibiotic susceptibility testing based on stress-activation of biosynthetic pathways that are the primary targets of antibiotics. We chose Staphylococcus aureus (S. aureus) as a model system due to its clinical importance, and we selected bacterial cell wall biosynthesis as the primary target of both stress and antibiotic. Enzymatic and mechanical stresses were used to damage the bacterial cell wall, and a β-lactam antibiotic interfered with the repair process, resulting in rapid cell death of strains that harbor no resistance mechanism. In contrast, resistant bacteria remained viable under the assay conditions. Bacteria, covalently-bound to the bottom of the microfluidic channel, were subjected to mechanical shear stress created by flowing culture media through the microfluidic channel and to enzymatic stress with sub-inhibitory concentrations of the bactericidal agent lysostaphin. Bacterial cell death was monitored via fluorescence using the Sytox Green dead cell stain, and rates of killing were measured for the bacterial samples in the presence and absence of oxacillin. Using model susceptible (Sanger 476) and resistant (MW2) S. aureus strains, a metric was established to separate susceptible and resistant staphylococci based on normalized fluorescence values after 60 min of exposure to stress and antibiotic. Because this ground-breaking approach is not based on standard methodology, it circumvents the need for minimum inhibitory concentration (MIC) measurements and long wait times. We demonstrate the successful development of a rapid microfluidic-based and stress-activated antibiotic susceptibility test by correctly designating the phenotypes of 16 additional clinically relevant S. aureus strains in a blinded study. In addition to future clinical utility, this method has great potential for studying the effects of various stresses on bacteria and their antibiotic susceptibility.