Ultrasound Characterization of Microbead and Cell Suspensions by Speed of Sound Measurements of Neutrally Buoyant Samples.

We present an experimental method including error analysis for the measurement of the density and compressibility of cells and microbeads; these being the two central material properties in ultrasound-based acoustophoretic applications such as particle separation, trapping, and up-concentration. The density of the microparticles is determined by using a neutrally buoyant selection process that involves centrifuging of microparticles suspended in different density solutions, CsCl for microbeads and Percoll for cells. The speed of sound at 3 MHz in the neutrally buoyant suspensions is measured as a function of the microparticle volume fraction, and from this the compressibility of the microparticles is inferred. Finally, from the obtained compressibility and density, the acoustic scattering coefficients and contrast factor of the microparticles are determined, and in a sensitivity analysis, the impact of the measurement errors on the computed acoustic properties is reported. The determination of these parameters and their uncertainties allow for accurate predictions of the acoustophoretic response of the microparticles. The method is validated by determining the density (0.1-1% relative uncertainty) and compressibility (1-3% relative uncertainty) of previously well-characterized polymer microbeads and subsequently applied to determine the density (0.1-1% relative uncertainty), compressibility (1% relative uncertainty), scattering coefficients, and acoustic contrast factors for nonfixed and fixed cells, such as red blood cells, white blood cells, DU-145 prostate cancer cells, MCF-7 breast cancer cells, and LU-HNSCC-25 head and neck squamous carcinoma cells in phosphate buffered saline. The results show agreement with published data obtained by other methods.

Elastomeric negative acoustic contrast particles for affinity capture assays.

This report describes the development of elastomeric capture microparticles (ECµPs) and their use with acoustophoretic separation to perform microparticle assays via flow cytometry.We have developed simple methods to form ECµPs by cross-linking droplets of common commercially available silicone precursors in suspension followed by surface functionalization with biomolecular recognition reagents. The ECµPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum, or diluted blood. In this study, these particles have been functionalized with antibodies to bind prostate specific antigen and immunoglobulin (IgG). Specific separation of the ECµPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast ECµPs at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast (ECµPs) and positive contrast particles (cells). Separated ECµPs were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.

Micropatterned avidin arrays on silicon substrates via photolithography, self-assembly and bioconjugation.

Processes for micropatterning protein arrays on inorganic substrates have gained attention in the development of biosensors and clinical diagnostics. This study demonstrates a chemically selective method based on photolithographic deposition of gold patterns with the subsequent attachment of functionalized alkanethiols via molecular self-assembly. Selective capping of carboxy groups on alkanethiols by N-hydroxysuccinimide esters allowed the deposition of methoxypoly(ethylene glycol) silane as a blocking agent on unpatterned regions. Carboxylates were used to couple a form of avidin to create a microarray of protein. This microarray was successfully probed with biotinylated quantum dots. In-process characterization methods included grazing-angle Fourier-transform IR spectroscopy, ellipsometry, contact-angle goniometry, atomic-force microscopy and fluorescence microscopy.