Fluorescent Lipo-Beads for the Sensitive Detection of Phospholipase A2 and Its Inhibitors.

Phospholipase A2 (PLA2) is a membrane lytic enzyme that is present in many organisms. Human PLA2 has emerged as a potential biomarker as well as a therapeutic target for several diseases including cancer, cardiovascular diseases, and some inflammatory diseases. The current study focuses on the development of lipo-beads that are very reactive and highly sensitive to PLA2. To develop the best supported lipid bilayer formulation, several lipid combinations were investigated using 10 µm porous silica beads. The reactivity of PLA2 was monitored via the decrease in particle fluorescence because of the release of entrapped fluorescent dye from the particle pores or the disintegration of a fluorescent lipid constituted on the bilayer upon lipid hydrolysis using flow cytometry. The enzyme binding studies indicate that lipo-beads with bulky fluorescent tags in the lipid head group and anionic lipids produce a more pronounced response. The kinetic studies suggest that these lipo-beads are very reactive with PLA2 and can generate a detectable signal in less than 5 min. The enzyme inhibition studies were also conducted with two known PLA2 inhibitors, varespladib and quercetin. We find that quercetin can hydrolyze the supported membrane, and thus inhibition of PLA2 is not observed; however, varespladib has shown significant PLA2 inhibition on lipo-beads. We have demonstrated that our lipo-bead-based approach can detect annexin-3, a known disease biomarker, as low as 10 nM within 5 min after incubation.

A toxicological study on photo-degradation products of environmental ibuprofen: Ecological and human health implications.

Increasing quantities of pharmaceutical waste in the environment have disrupted the balance of ecosystems, and may have subsequent effects on human health. Although a handful of previous studies have shown the impacts of pharmaceutically active compounds on the environment, the toxicological effects of their degradation products remain largely unknown. In the current study, the photo-degradation products of environmental ibuprofen were assessed for both ecotoxicological and human health effects using a series of in vitro assays. Here, six of the major degradation products are synthesized with high purity (>98%) and characterized with 1HNMR, 13CNMR, FT-IR and HRMS. To evaluate human health effects, three gut microbiota species, Lactobacillus acidophilus, Enterococcus faecalis and Escherichia coli, and two human cell lines, HEK293T and HepG2, are exposed to various concentrations of ibuprofen and its degradation products. On L. acidophilus, the ibuprofen degradation product (±)-(2R,3R)-2-(4-isobutylphenyl)-5-methylhexan-3-ol shows a greater toxic effect while ibuprofen enhances its growth at lower concentrations. At higher concentrations, ibuprofen shows at least a 2-fold higher toxicity compared to that of its degradation products. However, E. faecalis shows little or no effect upon exposure to these compounds. An induction of the SOS response in E. coli is observed but limited to only ibuprofen and 4-acetylbenzoic acid. In human cell line studies, survival of both HEK293T and HepG2 cell lines is profoundly impaired by the photo-degradation products of (±)- (2R,3R)-2-(4-isobutylphenyl)-5-methylhexan-3-ol, (±)-(2R,3S)-2-(4-isobutylphenyl)-5-methylhexan-3-ol, and (±)-1-(4-(1-hydroxy-2methylpropyl)phenyl)ethan-1-one. In this work, the bioluminescence bacterium, Aliivibrio fischeri, is used as a model to assess environmental impact. Both ibuprofen and its degradation products inhibit the growth of this gram-negative bacteria with the primary compound showing the most significant impact. Overall, our results highlight that some of the degradation products of ibuprofen can be more toxic to human kidney cell line and liver cell line than the parent compound while ibuprofen can be more toxic to human gut microbiota and A. fischeri than ibuprofen degradation products.

Separation of sub-micron particles from micron particles using acoustic fluid relocation combined with acoustophoresis.

Acoustophoresis has gained increasing attention as a gentle, non-contact, and high-throughput cell and particle separation technique. It is conveniently used to isolate and enrich particles that are greater than 2 µm; however, its use in manipulating particles smaller than 2 µm is limited. In this work, we present an alternative way of using acoustic forces to manipulate sub-micrometer particles in continuous flow fashion. It has been shown that acoustic forces can be employed to relocate parallel laminar flow streams of two impedance-mismatched fluids. We demonstrate the separation of sub-micron particles from micron particles by the combination of acoustophoresis and acoustic fluid relocation. The micron particles are focused into the middle of the flow channel via primary acoustic forces while sub-micron particles are moved to the side via drag forces created by the relocating fluid. We demonstrate the proof of the concept using binary mixtures of particles comprised of sub-micron/micron particles, micron/micron particles, and bovine red blood cells with E. coli. The efficiency of the particle enrichment is determined via flow cytometry analysis of the collected streams. This study demonstrates that by combining acoustic fluid relocation with acoustophoresis, sub-micron particles can be effectively separated from micron particles at high flow rates and it can be further implemented to separate binary mixtures of micron particles if the volumetric ratio of two particles is greater than 10 and the larger particle diameter is about 10 µm. The combined method is more appropriate to use than acoustophoresis in situations where acoustic streaming and differences in acoustic impedance of fluids can be of concern. Graphical abstract In the presence of a resonance acoustic field, the clean high-density fluid (dark gray) and the low-density sample fluid are relocated. During this process, E. coli are separated from the red blood cells (RBCs).

Simple and inexpensive micromachined aluminum microfluidic devices for acoustic focusing of particles and cells.

We introduce a new method to construct microfluidic devices especially useful for bulk acoustic wave (BAW)-based manipulation of cells and microparticles. To obtain efficient acoustic focusing, BAW devices require materials that have high acoustic impedance mismatch relative to the medium in which the cells/microparticles are suspended and materials with a high-quality factor. To date, silicon and glass have been the materials of choice for BAW-based acoustofluidic channel fabrication. Silicon- and glass-based fabrication is typically performed in clean room facilities, generates hazardous waste, and can take several hours to complete the microfabrication. To address some of the drawbacks in fabricating conventional BAW devices, we explored a new approach by micromachining microfluidic channels in aluminum substrates. Additionally, we demonstrate plasma bonding of poly(dimethylsiloxane) (PDMS) onto micromachined aluminum substrates. Our goal was to achieve an approach that is both low cost and effective in BAW applications. To this end, we micromachined aluminum 6061 plates and enclosed the systems with a thin PDMS cover layer. These aluminum/PDMS hybrid microfluidic devices use inexpensive materials and are simply constructed outside a clean room environment. Moreover, these devices demonstrate effectiveness in BAW applications as demonstrated by efficient acoustic focusing of polystyrene microspheres, bovine red blood cells, and Jurkat cells and the generation of multiple focused streams in flow-through systems. Graphical abstract The aluminum acoustofluidic device and the generation of multinode focusing of particles.

The intersection of flow cytometry with microfluidics and microfabrication.

A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry.

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.

One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry.

Multinode acoustic focusing for parallel flow cytometry.

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 µm, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multinode acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 µm in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of a CD4+ cellular immunophenotyping assay. This approach will have significant impact toward the creation of high throughput flow cytometers for rare cell detection applications (e.g., circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry.

Magnetic microsphere-based methods to study the interaction of teicoplanin with peptides and bacteria.

Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal D-Ala-D-Ala intermediates in the peptidoglycan of the cell wall of gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two D-Ala-D-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled D-Ala-D-Ala-D-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 x 10(-6) and 3.0 x 10(-6) mol L(-1), respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide-antibiotic interaction.

Detection of membrane biointeractions based on fluorescence superquenching.

Assays for biointeractions of molecules with supported lipid bilayers using fluorescence superquenching are described. A conjugated cationic polymer was adsorbed on to silica microspheres, which were then coated with an anionic lipid bilayer. The lipid bilayer attenuated superquenching by acting as a barrier between the conjugated polymer and its quencher. Biointeractions of the lipid bilayer with a membrane lytic peptide, melittin, were detected and quantitated by superquenching of the conjugated polyelectrolyte in flow cytometric and microfluidic bioassays. A higher sensitivity for detecting melittin lysis of the lipid bilayer at lower concentrations and shorter times for melittin action was found using flow cytometry in this study in comparison to other existing methods. This study combined the sensitivity of superquenching and flow cytometry to detect biointeractions with a lipid bilayer, which serves as a platform for developing functional assays for sensor applications, lipid enzymology, and investigations of molecular interactions. In addition, this study demonstrated proof-of-concept for using superquenching detected as a result of lipid bilayer disruption in a microfluidic format.

Biosensors based on release of compounds upon disruption of lipid bilayers supported on porous microspheres.

The authors describe a biosensing concept based on the release of compounds, which are encapsulated within lipid-coated porous silica microspheres, by detergents and toxins that disrupt supported lipid bilayers (SLBs) on the microspheres. Suspension and microfluidic based methods have been developed to monitor the release of the encapsulated compounds in response to membrane disruption. The authors established that the SLBs on porous microspheres can endure experimental conditions necessary for their incorporation into packed microchannels while maintaining the bilayer integrity and functionality. Model compounds including a nonionic detergent (Triton X-100), a membrane active protein (alpha-hemolysin), and a membrane lytic antimicrobial peptide (melittin) were successfully utilized to interact with different formulations of SLBs on porous silica microspheres. The results demonstrate the stability of the SLBs on the microspheres for several weeks, and the feasibility of using this system to detect the release of fluorescent dyes as well as other molecular reporters. The latter were detected by their involvement in subsequent biospecific interactions that were detected by fluorescence. This study exemplifies proof of concept for developing new chemical and biochemical sensors and drug delivery systems based on the disruption of lipid membranes coating porous silica microspheres that encapsulate dyes or bioactive compounds.

Fabrication of fritless chromatographic microchips packed with conventional reversed-phase silica particles.

This paper describes the development and study of a disposable and inexpensive microfluidic chip, fabricated from poly(dimethylsiloxane) (PDMS) incorporating conventional chromatographic reversed-phase silica particles (C18) without the use of frits, permanent physical barriers, tapers, or restrictors. The packing of C18 modified silica particles into the microfluidic channels is made possible by the hydrophobic nature and excellent elasticity of PDMS. Keystone-, clamping-, and anchor-effects provide the stability and the compactness of the packing and attenuated wall-effects were observed.

Superquenching as a detector for microsphere-based flow cytometric assays.

BACKGROUND: Fluorescent conjugated polymers display high fluorescence quantum yields and enhanced sensitivity to quenching (superquenching) by oppositely charged quenchers through energy or electron transfer. Fluorescent polymers and their quenchers are used in bead-based biosensor applications where the polymers are coated on particles. In this work, we investigate a detection method that utilizes superquenching on microspheres, which can be used for flow cytometric assays. METHODS: Microspheres were coated with the fluorescent cationic polyelectrolyte poly(p-phenylene-ethynylene) (PPE), and its superquenching by 9,10-anthraquinone-2,6-disulfonic acid (AQS) was examined by fluorometric methods in presence and in absence of a barrier to superquenching in the form of an anionic lipid bilayer. RESULTS: Flow cytometry detected superquenching of PPE on microspheres (MS-PPE) by AQS where high levels of reduction in fluorescence were observed. Adding different concentrations of AQS to MS-PPE yielded a Stern-Volmer quenching constant of 0.8x10(6) M-1. While forming an anionic lipid bilayer around the MS-PPE acted as a barrier to superquenching by AQS, disrupting the lipid bilayer allowed superquenching to take place. CONCLUSIONS: The sensitivity of flow cytometry in detecting fluorescence of microspheres and the amplified quenching sensitivity of fluorescent conjugated polymers both offer advantages over other fluorometric methods and conventional quenching detection. This study used superquenching of fluorescent polymers as a new tool in flow cytometry, thus combining the advantages offered by both method and detector. In addition, we employed the formation and the disruption of a supported lipid bilayer in mediating superquenching to offer new biosensing applications.

Near-simultaneous and real-time detection of multiple analytes in affinity microcolumns.

A miniaturized immunoassay system based on beads in poly(dimethylsiloxane) microchannels for analyzing multiple analytes has been developed. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer. A preliminary account of this work based on single-analyte format has been published in this journal (Buranda, T.; Huang, J.; Perez-Luna, V. H.; Schreyer, B.; Sklar, L. A.; Lopez, G. P. Anal. Chem. 2002, 74, 1149-1156). We have extended the work to a multianalyte model system composed of discrete segments of beads that bear distinct receptors. Near-simultaneous and real-time detection of diverse analytes is demonstrated. The importance of this work is established in the exploration of important factors related to the design, assessment, and utility of affinity microcolumn sensors. First, beads derivatized with surface chemistry suitable for the attachment of fluorescently labeled biomolecules of interest are prepared and characterized in terms of functionality and receptor site densities by flow cytometry. Second, calibrated beads are incorporated in microfluidic channels. The analytical device that emerges replicates the basic elements of affinity chromatography with the advantages of microscale and real-time direct measurement of bound analyte on beads rather than the indirect determination from eluted sample typical of affinity chromatography. In addition, the two-compartment analysis of the assay data as demonstrated in single-analyte columns provides a template upon which the dynamics of multiple-analyte assays can be characterized using existing theoretical models and be tested experimentally. The assay can potentially detect subfemtomole quantities of protein with high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter to submicroliter volumes of analyte fluid. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes (e.g., biomolecular indicators of diseases). Proof-of-principle analytes include FLAG peptide and carcinoembryonic antigen detected at physiologically relevant concentration levels.