Unzipping Kinetics of Double-Stranded DNA in a Nanopore

We studied the unzipping kinetics of single molecules of double-stranded DNA by pulling one of their two strands through a narrow protein pore. PCR 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.Comment: 10 pages, 5 figures, Accepted: Physics Review Letter

Optimization of Stress-Based Microfluidic Testing for Methicillin Resistance in Staphylococcus aureus Strains

The rapid evolution of antibiotic resistance in bacterial pathogens is driving the development of innovative, rapid antibiotic susceptibility testing (AST) tools as a way to provide more targeted and timely antibiotic treatment. We have previously presented a stress-based microfluidic method for the rapid determination of antibiotic susceptibility in methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). In this method, stress is used to potentiate the action of antibiotics, and cell death is measured as a proxy for susceptibility. The method allows antibiotic susceptibility to be determined within an hour from the start of the antibiotic introduction. However, the relatively low dynamic range of the signal (2–10% cell response) even with high antibiotic concentrations (10–50 µg/mL) left room for the method’s optimization. We have conducted studies in which we varied the flow patterns, the media composition, and the antibiotic concentration to increase the cell death response and concordantly decrease the required antibiotic concentration down to 1–3 µg/mL, in accordance with the Clinical and Laboratory Standards Institute’s (CLSI) guidelines for AST breakpoint concentrations

Automated tissue dissociation for rapid extraction of viable cells

Viable cells from healthy tissues are a rich resource in high demand for many next-generation therapeutics and regenerative medicine applications. Cell extraction from the dense connective matrix of most tissues is a labor-intensive task and high variability makes cGMP compliance difficult. To reduce costs and ensure greater reproducibility, automated tissue dissociators compatible with robotic liquid handling systems are required. Here we demonstrate the utility of our automated tissue dissociator that is compatible with standard microtiter well plates for high-throughput processing. We show that viable cell yields match or exceed manual methods while reducing processing time by at least 85%

Rapid phenotypic stress-based microfluidic antibiotic susceptibility testing of Gram-negative clinical isolates

Bacteremia is a life-threatening condition for which antibiotics must be prescribed within hours of clinical diagnosis. Since the current gold standard for bacteremia diagnosis is based on conventional methods developed in the mid-1800s—growth on agar or in broth—identification and susceptibility profiling for both Gram-positive and Gram-negative bacterial species requires at least 48–72 h. Recent advancements in accelerated phenotypic antibiotic susceptibility testing have centered on the microscopic growth analysis of small bacterial populations. These approaches are still inherently limited by the bacterial growth rate. Our approach is fundamentally different. By applying environmental stress to bacteria in a microfluidic platform, we can correctly assign antibiotic susceptibility profiles of clinically relevant Gram-negative bacteria within two hours of antibiotic introduction rather than 8–24 h. The substantial expansion to include a number of clinical isolates of important Gram-negative species—Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa—reported here underscores the broad utility of our approach, complementing the method’s proven utility for Gram-positive bacteria. We also demonstrate that the platform is compatible with antibiotics that have varying mechanisms of action—meropenem, gentamicin, and ceftazidime—highlighting the versatility of this platform

Graph showing the percent recoveries of viable microorganisms following our sample preparation protocol.

<p>Data are averages of several replicates (n>3).</p

Results from PCR.

<p>A: PCR data show that pellets from whole blood samples (10 mL) spiked with higher concentrations of MSSA amplify sooner (pellet volume in PCR well = 8 μL). B: Contingency table for PCR analysis of processed positive (100 CFU/mL) and negative (0 CFU/mL) whole blood samples.</p

List of microorganisms used in the current study and their cultivation conditions.

<p>^<i>a</i> Gram-positive bacterium;</p><p>^<i>b</i> Gram-negative bacterium;</p><p>^<i>c</i> Yeast</p><p>List of microorganisms used in the current study and their cultivation conditions.</p

Images of the custom-made microbial concentration devices.

<p>Left: a blown-up view of the individual parts of the device. Right: a CAD model of a fully assembled device.</p

Images of the pellets after lysing 10 mL of whole blood.

<p>A: Microscopic view of an unspiked blood sample after lysis process at 60x magnification; B: For reference, microscopic view of unlysed whole blood at 60x magnification; C: Front view of the pellets; D: Side view of the pellets; E: For reference, from left to right, 2.5, 5.0, 10, and 15 μL of whole blood.</p