Daniel C. Leslie

New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, enabling label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without prior DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques.

Periodic response of fluidic networks with passive deformable features

This paper outlines the scaling parameters governing the frequency response of fluidic networks with embedded deformable features, which are subjected to periodic excitation. These parameters describe the impact of deformable feature properties on the relative importance of potential energy, kinetic energy, and viscous dissipation. They are used to identify device characteristics that produce specific frequency responses, such as low-pass, high-pass, and bandpass filters that exploit (or avoid) the effects of fluid inertia. Simulations illustrate that passive deformable diodes have little effect on the frequency response of high-pass filters comprised of elastomer features.

A simple method for the evaluation of microfluidic architecture using flow quantitation via a multiplexed fluidic resistance measurement

Quality control of microdevices adds significant costs, in time and money, to any fabrication process. A simple, rapid quantitative method for the post-fabrication characterization of microchannel architecture using the measurement of flow with volumes relevant to microfluidics is presented. By measuring the mass of a dye solution passed through the device, it circumvents traditional gravimetric and interface-tracking methods that suffer from variable evaporation rates and the increased error associated with smaller volumes. The multiplexed fluidic resistance (MFR) measurement method measures flow via stable visible-wavelength dyes, a standard spectrophotometer and common laboratory glassware. Individual dyes are used as molecular markers of flow for individual channels, and in channel architectures where multiple channels terminate at a common reservoir, spectral deconvolution reveals the individual flow contributions. On-chip, this method was found to maintain accurate flow measurement at lower flow rates than the gravimetric approach. Multiple dyes are shown to allow for independent measurement of multiple flows on the same device simultaneously. We demonstrate that this technique is applicable for measuring the fluidic resistance, which is dependent on channel dimensions, in four fluidically connected channels simultaneously, ultimately determining that one chip was partially collapsed and, therefore, unusable for its intended purpose. This method is thus shown to be widely useful in troubleshooting microfluidic flow characteristics.

Size‐based separations as an important discriminator in development of proximity ligation assays for protein or organism detection

Proximity ligation is a powerful technique to measure minute concentrations of target protein with high specificity, and it has been demonstrated to be effective on a wide variety of protein targets. The proximity ligation assay (PLA) technique is shown to be compromised by the amplification of a nonspecific fluorescent product that is not indicative of protein presence, which was previously unidentified in a published procedure. This result illuminates the complexity of designing the optimal PLA and the possibility of using a size‐based separation to increase the reliability of PLAs in general. Nucleic acid controls were developed to optimize the assay, which led to a novel end‐point detection method that exploits microchip electrophoresis to size the products. This method provides a greater ability to distinguish a between the target proteins signal and noise in a PLA. The utility of the PLA is demonstrated by the detection of human pathogenic Escherichia coli O157:H7 bacteria, a pathogen at the root of many recent life‐threatening food poisoning outbreaks. The results of the PLA show a detection limit of 100 E. coli O157:H7 cells with minimal cross‐reactivity with gram positive control Staphylococcus aureus bacteria. The advantages of miniaturizing this process are the 100‐fold reduction in volume, greatly reducing reagent requirements, and doubling of the thermocycling speed via noncontact infrared heating. This work, consequently, adds to the understanding of background fluorescence in PLAs, provides a method for evaluating nonspecific amplification, and shows that a qualitative PCR response indicative of the presence protein can be achieved with PLA.

A New Tool for Oligonulceotide Import into Cells

The field of gene therapy potentially offers physicians an entirely new set of armaments with which to battle disease. The approach, radical in concept but powerful in its potential effect, generically aims to correct defective genes responsible for disease development by inserting the normal gene into a nonspecific location in the genome or by swapping out the abnormal gene through homologous recombination. Perhaps more tangible, near-term approaches are those that look to either repair the abnormal gene via selective reverse mutation or “knockdown” the expression of the mutated gene. Delivery of oligonucleotides, whether as entire genes or shorter antisense strands, remains one of the most significant challenges in the field. The linchpin to success is the ability to efficiently and selectively insert oligonucleotides into the desired cells. Current efforts in the application of oligonucleotide delivery in this and other arenas are restricted by the relatively low efficiency of methods used for oligonucleotide transfer. In addition, potential hazards exist with any method that introduces foreign biological material into the body—toxicity issues as well as the potential for mounting an immune response against a specific gene delivery vehicle complicate the challenge. It is against this backdrop that a series of papers out of the Mirkin group at Northwestern University is significant(1)(2)(3)(4). Few disagree with the power that …