Fluidics cube for biosensor miniaturization.

To create a small, portable, fully automated biosensor, a compact means of fluid handling is required. We designed, manufactured, and tested a "fluidics cube" for such a purpose. This cube, made of thermoplastic, contains reservoirs and channels for liquid samples and reagents and operates without the use of any internal valves or meters; it is a passive fluid circuit that relies on pressure relief vents to control fluid movement. We demonstrate the ability of pressure relief vents to control fluid movement and show how to simply manufacture or modify the cube. Combined with the planar array biosensor developed at the Naval Research Laboratory, it brings us one step closer to realizing our goal of a handheld biosensor capable of analyzing multiple samples for multiple analytes.

High-resolution finite element models with tissue strength asymmetry accurately predict failure of trabecular bone.

The ability to predict trabecular failure using microstructure-based computational models would greatly facilitate study of trabecular structure-function relations, multiaxial strength, and tissue remodeling. We hypothesized that high-resolution finite element models of trabecular bone that include cortical-like strength asymmetry at the tissue level, could predict apparent level failure of trabecular bone for multiple loading modes. A bilinear constitutive model with asymmetric tissue yield strains in tension and compression was applied to simulate failure in high-resolution finite element models of seven bovine tibial specimens. Tissue modulus was reduced by 95% when tissue principal strains exceeded the tissue yield strains. Linear models were first calibrated for effective tissue modulus against specimen-specific experimental measures of apparent modulus, producing effective tissue moduli of (mean+/-S.D.) 18.7+/-3.4GPa. Next, a parameter study was performed on a single specimen to estimate the tissue level tensile and compressive yield strains. These values, 0.60% strain in tension and 1.01% strain in compression, were then used in non-linear analyses of all seven specimens to predict failure for apparent tensile, compressive, and shear loading. When compared to apparent yield properties previously measured for the same type of bone, the model predictions of both the stresses and strains at failure were not statistically different for any loading case (p>0.15). Use of symmetric tissue strengths could not match the experimental data. These findings establish that, once effective tissue modulus is calibrated and uniform but asymmetric tissue failure strains are used, the resulting models can capture the apparent strength behavior to an outstanding level of accuracy. As such, these computational models have reached a level of fidelity that qualifies them as surrogates for destructive mechanical testing of real specimens.

Array biosensor for detection of biohazards.

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.

Array biosensor for simultaneous identification of bacterial, viral, and protein analytes.

The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes. The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies. This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes. To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay. Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time. The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods. Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively. The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format.

An array immunosensor for simultaneous detection of clinical analytes.

A fluorescence-based immunosensor has been developed for simultaneous analysis of multiple samples. A patterned array of recognition elements immobilized on the surface of a planar waveguide is used to "capture" analyte present in samples; bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This immunosensor was used to detect physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D-dimer, a marker of sepsis and thrombotic disorders, in spiked clinical samples.

Array biosensor: optical and fluidics systems.

Optical and fluidics systems have been developed as central components for an automated array biosensor. Disposable planar waveguides are patterned with immobilized capture antibodies using a physically isolated patterning (PIP) method. The PIP method enables simultaneous deposition of several antibodies and completely circumvents cross-immobilization problems encountered with other array deposition processes. A multi-channel fluidics cell allows numerous assays to be performed on the patterned waveguide. The sensing arrays are optically interrogated using a diode laser with a tailored output to optimize coupling to and maximize excitation uniformity within the waveguide. A patterned cladding is employed to optically isolate the waveguide from perturbations induced by the permanently attached flow cells. Compact optics image the evanescently excited fluorescence onto a large area, cooled CCD array. The image data is processed and automated signal analysis corrects for local background and noise variations.