Joel P. Golden

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using Attenuated Total Reflectance Infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces over similar time scales to the SPRI experiments. Barnacles were allowed to grow through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance at ...

Microbial biofilms grown utilizing electrodes as metabolic electron acceptors or donors are a new class of biomaterials with distinct electronic properties. Here we report that electron transport through living electrode-grown Geobacter sulfurreducens biofilms is a thermally activated process with incoherent redox conductivity. The temperature dependency of this process is consistent with electron-transfer reactions involving hemes of c-type cytochromes known to play important roles in G. sulfurreducens extracellular electron transport. While incoherent redox conductivity is ubiquitous in biological systems at molecular-length scales, it is unprecedented over distances it appears to occur through living G. sulfurreducens biofilms, which can exceed 100 microns in thickness.

The recognition of atomically distinct surface features by adsorbed biomolecules is central to the formation of surface-templated peptide or protein nanostructures. On mineral surfaces such as calcite, biomolecular recognition of, and self-assembly on, distinct atomic kinks and steps could additionally orchestrate changes to the overall shape and symmetry of a bulk crystal. In this work, we show through in situ atomic force microscopy (AFM) experiments that an acidic 20 kDa cement protein from the barnacle Megabalanus rosa (MRCP20) binds specifically to step edge atoms on {101̅4} calcite surfaces, remains bound and further assembles over time to form one-dimensional nanofibrils. Protein nanofibrils are continuous and organized at the nanoscale, exhibiting striations with a period of ca. 45 nm. These fibrils, templated by surface steps of a preferred geometry, in turn selectively dissolve underlying calcite features displaying the same atomic arrangement. To demonstrate this, we expose the protein solution to bare and fibril-associated rhombohedral etch pits to reveal that nanofibrils accelerate only the movement of fibril-forming steps when compared to undecorated steps exposed to the same solution conditions. Calcite mineralized in the presence of MRCP20 results in asymmetric crystals defined by frustrated faces with shared mirror symmetry, suggesting a similar step-selective behavior by MRCP20 in crystal growth. As shown here, selective surface interactions with step edge atoms lead to a cooperative regime of calcite modification, where templated long-range protein nanostructures shape crystals.

Bacterial infection and intoxication can present with common symptoms. The ability to identify a bacteria or toxin rapidly in clinical samples is critical for administering the appropriate treatment. The microflow cytometer has previously demonstrated the ability to test for six bacteria and toxins simultaneously in buffer. In this study, the number of bacteria and toxins analyzed was increased to ten, positive and negative controls were incorporated in all assays, and most importantly, multiplexed immunoassays were demonstrated in clinical matrices. The multiplexed assays using the microflow cytometer demonstrated detection limits similar to or better than other reported antibody-based methods for pathogen detection (ELISA, lateral flow, array biosensors). In most cases, detection from complex clinical matrices (serum and nasal wash) achieved limits of detection equivalent to those for spiked buffer samples. Clinical samples spiked with bacteria and/or toxins were also analyzed successfully in blind trials.

An integrated system with automated immunomagnetic separation and processing of fluidic samples was demonstrated for multiplexed optical detection of bacterial targets. Mixtures of target-specific magnetic bead sets were processed in the NRL MagTrap with the aid of rotating magnet arrays that entrapped and moved the beads within the channel during reagent processing. Processing was performed in buffer and human serum matrixes with 10-fold dilutions in the range of 10²–10⁶ cells/mL of target bacteria. Reversal of magnets’ rotation post-processing released the beads back into the flow and moved them into the microflow cytometer for optical interrogation. Identification of the beads and the detection of PE fluorescence were performed simultaneously for multiplexed detection. Multiplexing was performed with specifically targeted bead sets to detect E. coli 0157.H7, Salmonella Common Structural Antigen, Listeria sp., and Shigella sp., dose–response curves were obtained, and limits of detection were calculated for each target in the buffer and clinical matrix. Additional tests demonstrated the potential for using the MagTrap to concentrate target from larger volumes of sample prior to the addition of assay reagents.

Analysis of the intrinsic scatter and fluorescence profiles of marine algae can be used for general classification of organisms based on cell size and fluorescence properties. We describe the design and fabrication of a Microflow Cytometer on a chip for characterization of phytoplankton. The Microflow Cytometer measured distinct side-scatter and fluorescence properties of Synechococcus sp., Nitzschia d., and Thalassiosira p. Measurements were confirmed using the benchtop Accuri C6 flow cytometer. The Microflow Cytometer proved sensitive enough to detect and characterize picoplankton with diameter approximately 1 mm and larger phytoplankton of up to 80 mm in length. The wide range in size discrimination coupled with detection of intrinsic fluorescent pigments suggests that this Microflow Cytometer will be able to distinguish different populations of phytoplankton on unmanned underwater vehicles. Reversing the orientation of the grooves in the channel walls returns the sample stream to its original unsheathed position allowing separation of the sample stream from the sheath streams and the recycling of the sheath fluid.

The control of hydrodynamic focusing in a microchannel has inspired new approaches for microfluidic mixing, separations, sensors, cell analysis, and microfabrication. Achieving a flat interface between the focusing and focused fluids is dependent on Reynolds number and device geometry, and many hydrodynamic focusing systems can benefit from this understanding. For applications where a specific cross-sectional shape is desired for the focused flow, advection generated by grooved structures in the channel walls can be used to define the shape of the focused flow. Relative flow rates of the focused flow and focusing streams can be manipulated to control the cross-sectional area of the focused flows. This paper discusses the principles for defining the shape of the interface between the focused and focusing fluids and provides examples from our lab that use hydrodynamic focusing for impedance-based sensors, flow cytometry, and microfabrication to illustrate the breadth of opportunities for introducing new capabilities into microfluidic systems. We evaluate each example for the advantages and limitations integral to utilization of hydrodynamic focusing for that particular application.