Integrated acoustic immunoaffinity-capture (IAI) platform for detection of PSA from whole blood samples.

On-chip detection of low abundant protein biomarkers is of interest to enable point-of-care diagnostics. Using a simple form of integration, we have realized an integrated microfluidic platform for the detection of prostate specific antigen (PSA), directly in anti-coagulated whole blood. We combine acoustophoresis-based separation of plasma from undiluted whole blood with a miniaturized immunoassay system in a polymer manifold, demonstrating improved assay speed on our Integrated Acoustic Immunoaffinity-capture (IAI) platform. The IAI platform separates plasma from undiluted whole blood by means of acoustophoresis and provides cell free plasma of clinical quality at a rate of 10 uL/min for an online immunoaffinity-capture of PSA on a porous silicon antibody microarray. The whole blood input (hematocrit 38-40%) rate was 50 μl min(-1) giving a plasma volume fraction yield of ≈33%. PSA was immunoaffinity-captured directly from spiked female whole blood samples at clinically significant levels of 1.7-100 ng ml(-1) within 15 min and was subsequently detected via fluorescence readout, showing a linear response over the entire range with a coefficient of variation of 13%

Evaluation of Multidrug Efflux Pump Inhibitors by a New Method Using Microfluidic Channels

Fluorescein-di-β-d-galactopyranoside (FDG), a fluorogenic compound, is hydrolyzed by β-galactosidase in the cytoplasm of Escherichia coli to produce a fluorescent dye, fluorescein. We found that both FDG and fluorescein were substrates of efflux pumps, and have developed a new method to evaluate efflux-inhibitory activities in E. coli using FDG and a microfluidic channel device. We used E. coli MG1655 wild-type, ΔacrB (ΔB), ΔtolC (ΔC) and ΔacrBΔtolC (ΔBC) harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of Pseudomonas aeruginosa. Two inhibitors, MexB-specific pyridopyrimidine (D13-9001) and non-specific Phe-Arg-β-naphthylamide (PAβN) were evaluated. The effects of inhibitors on pumps were observed using the microfluidic channel device under a fluorescence microscope. AcrAB-TolC and analogous pumps effectively prevented FDG influx in wild-type cells, resulting in no fluorescence. In contrast, ΔB or ΔC easily imported and hydrolyzed FDG to fluorescein, which was exported by residual pumps in ΔB. Consequently, fluorescent medium in ΔB and fluorescent cells of ΔC and ΔBC were observed in the microfluidic channels. D13-9001 substantially increased fluorescent cell number in ΔBC/pABM but not in ΔBC/pXYM. PAβN increased medium fluorescence in all strains, especially in the pump deletion mutants, and caused fluorescein accumulation to disappear in ΔC. The checkerboard method revealed that D13-9001 acts synergistically with aztreonam, ciprofloxacin, and erythromycin only against the MexAB-OprM producer (ΔBC/pABM), and PAβN acts synergistically, especially with erythromycin, in all strains including the pump deletion mutants. The results obtained from PAβN were similar to the results from membrane permeabilizer, polymyxin B or polymyxin B nonapeptide by concentration. The new method clarified that D13-9001 specifically inhibited MexAB-OprM in contrast to PAβN, which appeared to be a substrate of the pumps and permeabilized the membranes in E. coli

Label-free cell separation and sorting in microfluidic systems

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible

Improved acoustophoretic circulating tumor cell (CTC) separation for low target cell numbers in clinical volumes

We present an improved acoustophoretic system for circulating tumor cell separation from blood. The system is operated by pressure driven flow, including flow sensors and a feed-back loop for precise flow control. The pressure driven system provides a user interface that can be handled by a non-skilled operator, and most importantly full clinical samples of 7 mL can be processed in 70 minutes. The improved flow control enables recovery of rare tumor cell populations ≈10 cells/mL spiked in whole blood at a recovery close to 100%

Acoustic resonances in straight micro channels: beyond the 1Dapproximation, Lab Chip (2008). doi: 10.1039/b801028e. O. Manneberg et al

Acoustic actuation can be used to perform several tasks in microfluidic systems. In this paper, we investigate an acoustic separator through micro-PIV analysis in stop-flow mode and numerical simulations, and a good agreement between the two is found. Moreover, we demonstrate that it is not sufficient only to characterize devices in flow-through mode, since in these systems much different resonant patterns can result in similarly looking band formations. Furthermore, we conclude that extended 1D approximations of the acoustic radiation force are inadvisable, and instead, a 2D model is preferred. The results presented here provide valuable insight into the nature and functionality of acoustic microdevices, and should be useful in the interpretation and understanding of the same

Acoustic whole blood plasmapheresis chip for prostate specific antigen microarray diagnostics.

The generation of high quality plasma from whole blood is of major interest for many biomedical analyses and clinical diagnostic methods. However, it has proven to be a major challenge to make use of microfluidic separation devices to process fluids with high cell content, such as whole blood. Here, we report on an acoustophoresis based separation chip that prepares diagnostic plasma from whole blood linked to a clinical application. This acoustic separator has the capacity to sequentially remove enriched blood cells in multiple steps to yield high quality plasma of low cellular content. The generated plasma fulfills the standard requirements (<6.0 x 10(9) erythrocytes/L) recommended by the Council of Europe. Further, we successfully linked the plasmapheresis microchip to our previously developed porous silicon sandwich antibody microarray chip for prostate specific antigen (PSA) detection. PSA was detected by good linearity (R(2) > 0.99) in the generated plasma via fluorescence readout without any signal amplification at clinically relevant levels (0.19-21.8 ng/mL)

Acoustic whole blood plasmapheresis chip for prostate specific antigen microarray diagnostics.

The generation of high quality plasma from whole blood is of major interest for many biomedical analyses and clinical diagnostic methods. However, it has proven to be a major challenge to make use of microfluidic separation devices to process fluids with high cell content, such as whole blood. Here, we report on an acoustophoresis based separation chip that prepares diagnostic plasma from whole blood linked to a clinical application. This acoustic separator has the capacity to sequentially remove enriched blood cells in multiple steps to yield high quality plasma of low cellular content. The generated plasma fulfills the standard requirements (<6.0 x 10(9) erythrocytes/L) recommended by the Council of Europe. Further, we successfully linked the plasmapheresis microchip to our previously developed porous silicon sandwich antibody microarray chip for prostate specific antigen (PSA) detection. PSA was detected by good linearity (R(2) > 0.99) in the generated plasma via fluorescence readout without any signal amplification at clinically relevant levels (0.19-21.8 ng/mL)