Microfluidic Sensors with Impregnated Fluorophores for Simultaneous Imaging of Spatial Structure and Chemical Oxygen Gradients.

Interior surfaces of polystyrene microfluidic structures were impregnated with the oxygen sensing dye Pt(II) tetra(pentafluorophenyl)porphyrin (PtTFPP) using a solvent-induced fluorophore impregnation (SIFI) method. Using this technique, microfluidic oxygen sensors are obtained that enable simultaneous imaging of both chemical oxygen gradients and the physical structure of the microfluidic interior. A gentle method of fluorophore impregnation using acetonitrile solutions of PtTFPP at 50 °C was developed leading to a 10-µm-deep region containing fluorophore. This region is localized at the surface to sense oxygen in the interior fluid during use. Regions of the device that do not contact the interior fluid pathways lack fluorophores and are dark in fluorescent imaging. The technique was demonstrated on straight microchannel and pore network devices, the latter having pillars of 300 µm diameter spaced center to center at 340 µm providing pore throats of 40 µm. Sensing within channels or pores and imaging across the pore network devices were performed using a Lambert LIFA-P frequency domain fluorescence lifetime imaging system on a Leica microscope platform. Calibrations of different devices prepared by the SIFI method were indistinguishable. Gradient imaging showed fluorescent regions corresponding to the fluid pore network, dark pillars, and fluorescent lifetime varying across the gradient, thus providing both physical and chemical imaging. More generally, the SIFI technique can impregnate the interior surfaces of other polystyrene containers, such as cuvettes or cell and tissue culture containers, to enable sensing of interior conditions.

Renewable surface fluorescence sandwich immunoassay biosensor for rapid sensitive botulinum toxin detection in an automated fluidic format.

A renewable surface biosensor for rapid detection of botulinum neurotoxin serotype A is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant protein fragment of the toxin heavy chain ( approximately 50 kDa) as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate non-overlapping epitopes of the full botulinum holotoxin ( approximately 150 kDa). Both of the targeted epitopes are located on the recombinant fragment. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by a sequential injection flow system, creating a 3.6 microL column. After perfusing the bead column with sample and washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degrees angle to one another delivered excitation light from a HeNe laser (633 nm) using one fiber and collected fluorescent emission light for detection with the other. After each measurement, the used Sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes using this system.

Lateral shearing interferometer for measuring photoinduced refractive index change in As(2)S(3).

We have built and demonstrated a lateral shearing interferometer as a process engineering and control tool for the fabrication and characterization of direct-laser-written waveguide structures in chalcogenide glasses. Photoinduced change in refractive index of 0.154+/-0.002 was measured for as-deposited amorphous As(2)S(3) thin films at 633 nm with an estimated measurement uncertainty of 1.3% for this air-gap interferometer configuration. The simple design of this interferometer can easily be adapted to other wavelengths including mid- and long-wave infrared regions to measure changes in refractive index or material inhomogeneities in transmissive materials.

Single-mode low-loss chalcogenide glass waveguides for the mid-infrared.

We demonstrate the design, fabrication, and characterization of single-mode low-loss waveguides for mid-infrared (MIR) wavelengths. Planar waveguide structures were fabricated from multilayer thin films of arsenic-based chalcogenide glasses followed by the creation of channel waveguides by using the photodarkening effect. Propagation losses as low as 0.5 dB/cm were measured for a quantum cascade laser end-fire coupled into the waveguides. This is a first step toward the design and fabrication of integrated optical components for MIR applications.

Single-walled carbon nanotube paper as a sorbent for organic vapor preconcentration.

Single-walled carbon nanotubes were examined as an adsorptive material for a thermally desorbed preconcentrator for organic vapors. The nanotubes were processed into a paper form and packed into a metal tube for flow-through sampling. Adsorbed vapors were released by a temperature-programmed desorption method and detected downstream with a flexural plate wave vapor sensor. The tested vapors, methyl ethyl ketone, toluene, and dimethyl methylphosphonate, were released from the packed column at different temperatures. The vapors were retained more strongly than previously observed for the widely used Tenax porous polymer, indicating a significant affinity of the single walled nanotubes for organic vapors.

Enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies.

Two immunoassay platforms were developed for either the sensitive or rapid detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. These antibodies also bind the same epitopes of the receptor binding domain present on a nontoxic recombinant heavy chain fragment used for assay development and testing in the current study. An enzyme-linked immunosorbent assay (ELISA) microarray using tyramide amplification for localized labeling was developed for the specific and sensitive detection of BoNT. This assay has the sensitivity to detect BoNT in buffer and blood plasma samples down to 14fM (1.4 pg mL(-1)). Three capture antibodies and one antibody combination were compared in the development of this assay. Using a selected pair from the same set of recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days. The renewable surface assay is less sensitive but much faster, providing results in less than 10 min.

Progressive thermal desorption of vapor mixtures from a preconcentrator with a porous metal foam internal architecture and variable thermal ramp rates.

A vapor preconcentrator has been designed with the porous polymer (Tenax) packed into a highly porous metal foam to facilitate thermal conductivity and temperature uniformity throughout the bed of the preconcentrator during heating. Vapors were desorbed using linear temperature programming from room temperature to a maximum temperature of 170 or 200 degrees C; the programmed duration of the thermal ramp was varied from 10 to 180 s. The partial separation of vapor mixtures that are thermally desorbed from the preconcentrator has been examined in terms of a metric for resolution, using methyl ethyl ketone, toluene, and dimethyl methylphosphonate as a test mixture. Vapors desorbed as a sequence of partially separated overlapping peaks, as observed with a polymer-coated flexural plate wave sensor. It was shown that vapor mixture resolution improved as the total time of the thermal ramp was extended from 30 to 120 s. In this way, the preconcentrator serves to act as a preseparator in addition to its usual functions for sampling, signal modulation, and improving sensitivity. Overlapping peaks were modeled, and peak areas were extracted using an exponentially modified Gaussian model. Peak areas were independent of the thermal ramp rate. Uses of such preconcentrators with multivariate detectors, such as sensor arrays, are discussed.