Buffer Flow Research Articles

Development of a Sequential Injection Analysis System for the Determination of Saccharin.

Saccharin is a powerfully sweet nonnutritive sweetener that has been approved for food-processing applications within the range of 100–1200 mg/kg. A simple, rapid, and cost-effective sequential injection analysis (SIA) technique was developed to determine the saccharin level. This method is based on the reaction of saccharin with p-chloranil in an ethanol medium with a hydrogen peroxide (H2O2) acceleration, and the resultant violet-red compound was detected using a UV-Vis spectrophotometer at λmax = 420 nm. To ascertain the optimal conditions for the SIA system, several parameters were investigated, including buffer flow rate and volume, p-chloranil concentration, and reactant volumes (saccharin, p-chloranil, and H2O2). The optimum setup of the SIA system was achieved with a buffer flow rate, buffer volume, and draw-up time of 1.2 mL/min, 2900 µL, and ~145 s, respectively. The optimal p-chloranil concentration is 30 mM, and the best reactant volumes, presented in an ordered sequence, are as follows: 30 µL of H2O2, 450 µL of saccharin, and 150 µL of p-chloranil. The optimized SIA configuration produced a good linear calibration curve with a correlation coefficient (R2 = 0.9812) in the concentration range of 20–140 mg/L and with a detection limit of 19.69 mg/L. Analytical applications in different food categories also showed acceptable recovery values in the range of 93.1–111.5%. This simple and rapid SIA system offers great feasibility for the saccharin quality control in food-product processing.

Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires.

Analyzing microRNAs (miRNAs) within urine extracellular vesicles (EVs) is important for realizing miRNA-based, simple, and noninvasive early disease diagnoses and timely medical checkups. However, the inherent difficulty in collecting dilute concentrations of EVs (<0.01 volume %) from urine has hindered the development of these diagnoses and medical checkups. We propose a device composed of nanowires anchored into a microfluidic substrate. This device enables EV collections at high efficiency and in situ extractions of various miRNAs of different sequences (around 1000 types) that significantly exceed the number of species being extracted by the conventional ultracentrifugation method. The mechanical stability of nanowires anchored into substrates during buffer flow and the electrostatic collection of EVs onto the nanowires are the two key mechanisms that ensure the success of the proposed device. In addition, we use our methodology to identify urinary miRNAs that could potentially serve as biomarkers for cancer not only for urologic malignancies (bladder and prostate) but also for nonurologic ones (lung, pancreas, and liver). The present device concept will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical checkups for cancer.

Insights into head-column field-amplified sample stacking: Part II. Study of the behavior of the electrophoretic system after electrokinetic injection of cationic compounds across a short water plug

Part I on head-column field-amplified sample stacking comprised a detailed study of the electrokinetic injection of a weak base across a short water plug into a phosphate buffer at low pH. The water plug is converted into a low conductive acidic zone and cationic analytes become stacked at the interface between this and a newly formed phosphoric acid zone. The fundamentals of electrokinetic processes occurring thereafter were studied experimentally and with computer simulation and are presented as part II. The configuration analyzed represents a discontinuous buffer system. Computer simulation revealed that the phosphoric acid zone at the plug-buffer interface becomes converted into a migrating phosphate buffer plug which corresponds to the cationically migrating system zone of the phosphate buffer system. Its mobility is higher than that of the analytes such that they migrate behind the system zone in a phosphate buffer comparable to the applied background electrolyte. The temporal behaviour of the current and the conductivity across the water plug were monitored and found to reflect the changes in the low conductivity plug. Determination of the buffer flow in the capillary revealed increased pumping caused by the mismatch of electroosmosis within the low conductivity plug and the buffer. This effect becomes elevated with increasing water plug length. For plug lengths up to 1% of the total column length the flow quickly drops to the electroosmotic flow of the buffer and simulations with experimentally determined current and flow values predict negligible band dispersion and no loss of resolution for both low and large molecular mass components.

Optimization of the linear quantification range of an online trypsin digestion coupled liquid chromatography–tandem mass spectrometry (LC–MS/MS) platform

ABSTRACTTandem mass spectrometry (MS/MS)-based proteomic workflows with a bottom-up approach require enzymatic digestion of proteins to peptide analytes, usually by trypsin. Online coupling of trypsin digestion of proteins, using an immobilized enzyme reactor (IMER), with liquid chromatography (LC) and MS/MS is becoming a frequently used approach. However, finding IMER digestion conditions that allow quantitative analysis of multiple proteins with wide range of endogenous concentration requires optimization of multiple interactive parameters: digestion buffer flow rate, injection volume, sample dilution, and surfactant type/concentration. In this report, we present a design of experiment approach for the optimization of an integrated IMER-LC–MS/MS platform. With bovine serum albumin as a model protein, the digestion efficacy and digestion rate were monitored based on LC–MS/MS peak area count versus protein concentration regression. The optimal parameters were determined through multivariate surface response modeling and consideration of diffusion controlled immobilized enzyme kinetics. The results may provide guidance to other users for the development of quantitative IMER-LC–MS/MS methods for other proteins.

Aptamer based surface plasmon resonance sensor for aflatoxin B1

The authors describe a surface plasmon resonance (SPR) based aptasensor for the carcinogenic mycotoxin aflatoxin B1 (AFB1) in a direct assay format. The aptamer is immobilized on the surface of a commercial sensor chip, and the SPR signal increases on binding of AFB1. The sensor chip can be fully regenerated by passing a flow of buffer over it upon which bound AFB1 dissociates from the aptamer. The biosensor works in the 0.4 nM to 200 nM AFB1 concentration range and has a 0.4 nM detection limit. It allows AFB1 to be determined in complex samples such as diluted red wine and beer. The assay is sensitive, and the chip is easily regenerated and stable. The method therefore overcomes certain limitations of antibody-based SPR assays and of competitive SPR assays for AFB1.

Effect of surface chemistry on tropomyosin binding to actin filaments on surfaces.

Reconstitution of actin filaments on surfaces for observation of filament-associated protein dynamics by fluorescence microscopy is currently an exciting field in biophysics. Here we examine the effects of attaching actin filaments to surfaces on the binding and dissociation kinetics of a fluorescence-labeled tropomyosin, a rod-shaped protein that forms continuous strands wrapping around the actin filament. Two attachment modalities of the actin to the surface are explored: where the actin filament is attached to the surface at multiple points along its length; and where the actin filament is attached at one end and aligned parallel to the surface by buffer flow. To facilitate analysis of actin-binding protein dynamics, we have developed a software tool for the viewing, tracing and analysis of filaments and co-localized species in noisy fluorescence timelapse images. Our analysis shows that the interaction of tropomyosin with actin filaments is similar for both attachment modalities. © 2016 Wiley Periodicals, Inc.

Simulation and experimental study of enzyme and reactant mixing in capillary electrophoresis based on-line methods

The establishment of an efficient reaction mixture represents a crucial part of capillary electrophoresis based on-line enzymatic assays. For ketamine N-demethylation to norketamine mediated by the cytochrome P450 3A4 enzyme, mixing of enzyme and reactants in the incubation buffer at physiological pH was studied by computer simulation. A dynamic electrophoretic simulator that encompasses Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure driven flow was utilized. The simulator in the diffusion mode was used to predict transverse diffusional reactant mixing occurring during hydrodynamic plug injection of configurations featuring four and seven plugs. The same simulator in the electrophoretic mode was applied to study electrophoretic reactant mixing caused by voltage application in absence of buffer flow. Resulting conclusions were experimentally verified with enantioselective analysis of norketamine in a background electrolyte at low pH. Furthermore, simulations visualize buffer changes that occur upon power application between incubation buffer and background electrolyte and have an influence on the reaction mixture.

Nanofluidic Fluorescence Microscopy (NFM) for real-time monitoring of protein binding kinetics and affinity studies

Kinetic monitoring of protein interactions offers insights to their corresponding functions in cellular processes. Surface plasmon resonance (SPR) is the current standard tool used for label-free kinetic assays; however, costly and sophisticated setups are required, decreasing its accessibility to research laboratories. We present a cost-effective nanofluidic-based immunosensor for low-noise real-time kinetic measurement of fluorescent-labeled protein binding. With the combination of fluorescence microscopy and reversed buffer flow operation, association and dissociation kinetics can be accessed in one single experiment without extra buffer loading step, which results in a simplified operation and reduced time of analysis compared to typical microfluidic immunoassays. Kinetic constants of two representative protein-ligand binding pairs (streptavidin/biotin; IgG/anti-IgG) were quantified. The good agreement of extracted rate constants with literature values and analogous SPR measurements indicates that this approach is applicable to study protein interactions of medium- and high-affinities with a limit of detection down to 1 pM, regardless of the analyte size.

Abstract 3956: DLD microfluidic purification and characterization of intact and viable circulating tumor cells in peripheral blood

Abstract We have developed a microfluidic chip-to-chip approach to purify circulating tumor cells (CTC’s). The first, a DLD (deterministic lateral displacement) microchip contains an array of microposts arranged in sub-arrays of different gap sizes. The “product” outlet of the DLD chip is connected with a second magnetic-separation chip. In the DLD chip, cells and particles are deflected or “bumped” based on their size, whereas the second chip is designed to remove any cell or component tagged with a magnetic particle (MNP). Air pressure is applied to maintain a constant flow of sample and buffer throughout the system in a vertical fashion. First, whole blood is mixed with biotinylated antibodies to CD45 and strepavidin-MNP's for 20min, diluted in buffer and passed through the DLD chip where the blood interacts with the posts and cells are bumped based on a deterministic lateral displacement principle, thus separating blood components. The “product” fraction from the DLD chip containing white blood cells and other larger cells is directed to the magnetic chip and the “waste” fraction containing red blood cells, platelets, debris and soluble blood components is discarded. The second chip has an array of magnets, capturing the sav-MNP/CD45+-tagged cells thereby allowing the gentle free flow of rare cells towards the final product fraction. With the chip-to-chip configuration we are able to process up to 8ml of blood, removing &amp;gt;99.8% of red blood cells and &amp;gt;98% of white blood cells, with the remaining purified cell populations suitable for further analysis and characterization. Validation of the chip-to-chip approach using tumor-derived cancer cells, including MDA-MB-231, PC-3 and SKBR-3 cells, confirms a linear recovery of &amp;gt;80% of the spiked-cells with a viability of ∼90%. The minimum cell size captured by our approach is approximately 6μm ensuring the isolation of small-sized CTC’s. Multi-color flow cytometry and imaging analysis of the chip-to-chip isolated cells from blood of breast cancer patients confirms the purification and identification of unique populations of HER2+/CD45-/EpCAM+ and CD146+/CD44+ cells characteristic of breast carcinomas-among other rare cell populations. Our chip-to-chip approach allows a gentle purification of intact and viable rare cells from cancer patients’ blood, suitable for functional analysis, drug sensitivity tests and genetic characterization. Thus, we have developed a hands-free liquid biopsy capable of isolating and purifying rare circulating tumor cells. Citation Format: Myra Koesdjojo, Zendra Lee, Christopher Dosier, Tanisha Saini, Khushroo Gandhi, Alison Skelley, Lee Aurich, Gregory Yang, Tony Ward, Roberto Campos-González. DLD microfluidic purification and characterization of intact and viable circulating tumor cells in peripheral blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3956.

Realization of Efficient High Throughput Buffering Policies for Network on Chip Router

The communication between processing elements is suffering challenges due to power, area and latency. Temporary flit storage during communication consumes the maximum power of the whole power consumption of the chip. The majority of current NoCs consume a high amount of power and area for router buffers only. Removing buffers and virtual channels (VCs) significantly simplifies router design and reduces the power dissipation by a considerable amount. The buffering scheme used in virtual channeling in a network- on-chip based router plays a significant role in determining the performance of the whole network-on- chip based mesh. Elastic buffer (EB) flow control is a simple control logic in the channels to use pipeline flip- flops (FFs) as storage locations. With the use of elastic buffers, input buffers are no longer required hence leading to a simplified router design. In this paper properties of buffers are studied with a test microarchitecture router for several packet injection rates given at an input port. The prime contribution of this article is the evaluation of various forms of the elastic buffers for throughput, FPGA resource utilization, average power consumed, and the maximum speed offered. The article also gives a comparison with some available buffering policies against throughput. The paper presents the synthesis and implementation on FPGA platforms. The work will help NoC designers in suitable simple router implementation for their FPGA design. The implementation targets Virtex5 FPGA and Stratix III device family. Index Terms—Network-on-chip, virtual channels, buffers.

Characterization of a miniaturized liquid bridge for nL sample infusion: a comparative study of sample flush-out behavior using flow simulations and direct ESI-MS analysis

In this work we shed light on the microfluidics of a miniaturized liquid bridge that forms the central part of a so-called “capillary gap sampler,” a novel device for rapid and seamless injection of nanoliter sample volumes into an electrospray ionization mass spectrometer (ESI-MS). Parameters relevant for sample flush-out at the liquid bridge and in the spray capillary were identified by systematic variation of the capillary dimensions, the linear buffer flow rate (2.1–34 mm/s) and molecular weight of the analytes (0.5–30 kDa). We found that a reduction in capillary wall thickness by a factor of 1.6 significantly influences analyte peak shapes, leads to an inversion of the relationship between peak width and analyte molecular weight, and allows a fivefold decrease in peak width for large molecules down to 5 s. The results could be verified and explained by simulations, in which the presence of diffusion-controlled “dead zones” at the liquid bridge and dispersion in the spray tip that depend on analyte molecular weight were identified as key factors relevant for the sample flush-out process. The merging of simulations and experimental data gives useful hints toward the re-design of a spray tip as built-in ESI-MS interface for an optimized gap sampler performance.

Swift and Energy Efficient Big Data Gathering Approaches in Wireless Sensor Networks

In Recent studies, mobile element acts as a mechanical carrier equipped with a powerful transceiver and battery. It directly collects the data from the sensors in the sensing environment via single-hop communication when traversing its transmission range and eventually delivers the collected data to the remote central. As a mobile element collects the data from every sensor node, the length of the mobile element tour will be increased. It results in increased data gathering latency. To solve this problem, several algorithms have been proposed. One of them called Toward Energy Efficient Big Data Gathering (TEEBD). Even it simplifies the mobile element data gathering by calculating the optimum number of clusters. Mobile element should wait until all of its cluster members uploads its data. It gives increased data gathering latency, and Packet loss due to buffer flow. In this paper, we propose two novel approaches called Energy Efficient Big Data Gathering using Local data Collector (EEBDG-LC) and Energy Efficient Big Data Gathering using Local data Collector with Threshold (EEBDG-LCWT). First approach concentrates on placing a local data collector in every centroid of the region. In which mobile element collects the information only from local data collector instead of all of its sensor nodes. It increases the speed of mobile element data gathering. The main goal of the second approach is to reduce the traffic in the local sensing region of EEBDG-LC based on the threshold value. In which node reaches the threshold value are only allowed to transmit data to the local data collector. Others go to the sleep mode immediately. Thus, increases the lifetime of the sensor network, and packet delivery ratio. Various data gathering mechanisms such as mobile element data gathering and data gathering using UAV have been used and comparison between these two has been done. The effectiveness of our approach is validated through extensive simulations.

Development and validation of a novel RP-HPLC method for stability-indicating assay of Abiraterone acetate

ABSTRACTAbiraterone acetate is a prodrug of Abiraterone widely used for the treatment of metastatic castration resistant prostate cancer. In this study, a simple, sensitive, and rapid stability-indicating reverse phase HPLC method was developed and validated for the determination of Abiraterone acetate in bulk and its pharmaceutical formulation. The method was developed by HPLC using a Hypersil ODS C-18 (150 mm × 4.6 mm, 5 µm) column in a isocratic mode with mobile phase constituted by potassium phosphate buffer and acetonitrile (40:60, v/v%) flow rate was 1.0 mL min−1, column temperature of 30°C, UV detection wavelength 235 nm, and injection volume of 20 µL. The validated parameters were in accordance with FDA and ICH specifications, assay exhibited a linear range of 25–250 µg mL−1 with regression (r2) coefficient 0.9998. The limits of detection and quantification were 0.23 and 0.70 µg mL. Accuracy was between 99.34 and 100.07%. The drug was subjected to various stress conditions like acidic, base hydrolysis, oxidation, thermal, and photolytic degradation. Stress study Abiraterone acetate was found susceptible to degrade under hydrolytic (acid and base) conditions. The proposed method has stability indicating the resolution of the main peak from their degradation peaks. The validated method is suitable for quality control application and reduced analysis time.

Utilizing a Key Aptamer Structure-Switching Mechanism for the Ultrahigh Frequency Detection of Cocaine.

Aptasensing of small molecules remains a challenge as detection often requires the use of labels or signal amplification methodologies, resulting in both difficult-to-prepare sensor platforms and multistep, complex assays. Furthermore, many aptasensors rely on the binding mechanism or structural changes associated with target capture by the aptameric probe, resulting in a detection scheme customized to each aptamer. It is in this context that we report herein a sensitive cocaine aptasensor that offers both real-time and label-free measurement capabilities. Detection relies on the electromagnetic piezoelectric acoustic sensor (EMPAS) platform. The sensing interface consists of a S-(11-trichlorosilyl-undecanyl)benzenethiosulfonate (BTS) adlayer-coated quartz disc onto which a structure-switching cocaine aptamer (MN6) is immobilized, completing the preparation of the MN6 cocaine aptasensor (M6CA). The EMPAS system has recently been employed as the foundation of a cocaine aptasensor based on a structurally rigid cocaine aptamer variant (MN4), an aptasensor referred to by analogy as M4CA. M6CA represents a significant increase in terms of analytical performance, compared to not only M4CA but also other cocaine aptamer-based sensors that do not rely on signal amplification, producing an apparent K(d) of 27 ± 6 μM and a 0.3 μM detection limit. Remarkably, the latter is in the range of that achieved by cocaine aptasensors relying on signal amplification. Furthermore, M6CA proved to be capable not only of regaining its cocaine-binding ability via simple buffer flow over the sensing interface (i.e., without the necessity to implement an additional regeneration step, such as in the case of M4CA), but also of detecting cocaine in a multicomponent matrix possessing potentially assay-interfering species. Finally, through observation of the distinct shape of its response profiles to cocaine injection, demonstration was made that the EMPAS system in practice offers the possibility to distinguish between the binding mechanisms of structure-switching (MN6) vs rigid (MN4) aptameric probes, an ability that could allow the EMPAS to provide a more universal aptasensing platform than what is ordinarily observed in the literature.

Insulin loaded mucus permeating nanoparticles: Addressing the surface characteristics as feature to improve mucus permeation

AimIt was the aim of this study to combine two strategies – namely the virus-mimicking strategy and the surface PEGylation strategy – in order to generate highly mucus permeating nanocarriers for oral insulin delivery. MethodsChondroitin sulphate was covalently conjugated with poly(ethylene glycol) 5kDa at different degree of modification and with the functionalized polymers NPs were formulated via complexation with chitosan. NPs were characterized by particle size, zeta potential, surface hydrophilicity and permeation ability in porcine mucus and on excised mucosa. ResultsThe NPs presented a size between 510 and 670nm and a zeta potential between −1 and −5mV when dispersed in simulated intestinal fluid. The mucus permeation test revealed a correlation between the NPs hydrophilicity and their ability to move through mucus. A 5-fold higher amount of NPs with the higher degree of PEGylation could permeate through fresh mucus compared to non-PEGylated NPs. Moreover, highly PEGylated NPs showed a 3.7-fold greater ability to be retained in intestinal mucosa against buffer flow compared to unmodified NPs. Finally, insulin was incorporated with a payload of 2.18% and the release profile showed a 65% release within 4h. ConclusionsResults of this study provide strong evidence for the potential of combining different surface modification strategies in order to improve the mucus permeating properties of NPs for oral peptide delivery.

Effects of Timber Harvest on Water Quantity and Quality in Small Watersheds in the Piedmont of North Carolina

This paired watershed study tested the effects of timber harvest on water quantity and quality in the North Carolina Piedmont physiographic region. Four headwater watersheds at Hill Demonstration Forest (HF1, HF2, HFW1, and HFW2) and two at Umstead Research Farm (UF1 and UF2) were continuously monitored for discharge and water quality from 2007 to 2013. The HF1 and UF1 watersheds were clearcut (treatment), leaving a 15.2-m vegetated riparian buffer around the streams to protect water quality as described in the North Carolina Neuse River Basin Riparian Buffer Rule. HF2 and UF2 were uncut and used as reference watersheds. Merchantable timber was selectively removed from the riparian buffer, reducing tree basal area by 27% in HF1 and 48% in UF1. HF1 and HF2 were nested within HFW1; thus, HFW1 was considered a partial cut where 33% of the watershed area was harvested, and HFW2 was the reference. We found that discharge in treatment watersheds increased dramatically, averaging 240% in HF1 and 200% in UF1 and 40% in HFW1 during the postharvest period, 2011–2013. Total suspended sediment export in the treatment watersheds also increased significantly in HF1 after harvest, probably due to the increase of discharge and movement of in-channel legacy sediment. Stormflow peak nitrate reached its maximum concentration during the first 2 years after harvest in the treatment watersheds and then declined, corresponding to the rapid regrowth of woody and herbaceous plants in the riparian buffer and uplands. We found that 36% of the UF1 streambank trees were blown down but did not cause a measurable increase in mean daily stormflow total suspended sediment concentration. Most buffer tree blowdown occurred during the first few years after a harvest. Bioclassification of benthic macroinvertebrates indicated that stream water quality remained good/fair to excellent in the treatment watersheds after the harvest. We conclude that the temporary increases in discharge were relatively large for the Piedmont region compared with those for other regions in the southeastern United States. However, the increases in channel sediment transport and nutrient exports associated with the hydrologic change did not have a measurable impact on the indicators of aquatic invertebrate community health or bioclassification rankings. Management and Policy Implications: There are three distinct land provinces across North Carolina: the mountains, Piedmont, and coastal plain. Understanding how region-specific watershed hydrology responds to land management and natural disturbances can provide useful information to land managers as they set flow targets needed to maintain ecological integrity in surface waters or to design riparian buffers for water quality protection. We found that after a clearcut timber harvest with a riparian buffer zone around the streams, the percent increases in annual discharge and mean nitrate concentrations tended to be higher in the Piedmont region than in the other two regions. Our study also found that tree blowdown in the riparian buffer was more likely to occur in the Piedmont Triassic region than in the Piedmont Carolina Slate Belt region. In the Triassic region, additional management activities should be part of the preharvest planning process as they may help mitigate windthrow and uprooting of streambanks and improve the overall riparian buffer functions and flow dynamics. For example, land planners or loggers should assess whether soils have Triassic or Slate Belt characteristics to determine if they need to refrain from creating gaps and retain more windfirm species within the riparian buffer. Although there was variation in blowdown occurrences among regions, Triassic soils comprise about 3.5% of North Carolina's total land area, so any statewide implications would be low and recommendations would be localized.

Microfluidic generation of hollow Ca-alginate microfibers

This work reports on an efficient microfluidic approach for continuous production of hollow Ca-alginate microfibers with controllable structures and functions. A coaxial microcapillary microfluidic device combined with a rotator is constructed to produce a cylindrical flow jet with four aqueous solutions as templates for continuous fabrication and collection of microfibers. A four-aqueous-phase flow jet with an intermediate buffer flow between the Ca(2+)-containing and alginate-containing flows is used as the template for microfiber fabrication. The buffer flow efficiently controls the diffusion of Ca(2+) into the alginate-containing flow as well as the crosslinking reaction, thus ensuring the continuous fabrication of hollow Ca-alginate microfibers under relatively low flow rates without clogging of the microchannel. The structure of the hollow microfibers can be flexibly adjusted by changing the flow rates and device dimensions. Meanwhile, the continuous fabrication process of the microfibers allows flexible incorporation of a functional component into the sheath flow for functionalization and addition of active substances in the core flow for encapsulation. This is demonstrated by fabricating hollow Ca-alginate microfibers with a wall containing magnetic nanoparticles for magnetic functionalization and with hollow internals containing Chlorella pyrenoidosa cells for confined growth. This work provides an efficient strategy for continuous fabrication of functional hollow Ca-alginate microfibers with controllable structures and functions.

Enhancement of microfluidic particle separation using cross-flow filters with hydrodynamic focusing.

A microfluidic chip is proposed to separate microparticles using cross-flow filtration enhanced with hydrodynamic focusing. By exploiting a buffer flow from the side, the microparticles in the sample flow are pushed on one side of the microchannels, lining up to pass through the filters. Meanwhile a larger pressure gradient in the filters is obtained to enhance separation efficiency. Compared with the traditional cross-flow filtration, our proposed mechanism has the buffer flow to create a moving virtual boundary for the sample flow to actively push all the particles to reach the filters for separation. It further allows higher flow rates. The device only requires soft lithograph fabrication to create microchannels and a novel pressurized bonding technique to make high-aspect-ratio filtration structures. A mixture of polystyrene microparticles with 2.7 μm and 10.6 μm diameters are successfully separated. 96.2 ± 2.8% of the large particle are recovered with a purity of 97.9 ± 0.5%, while 97.5 ± 0.4% of the small particle are depleted with a purity of 99.2 ± 0.4% at a sample throughput of 10 μl/min. The experiment is also conducted to show the feasibility of this mechanism to separate biological cells with the sample solutions of spiked PC3 cells in whole blood. By virtue of its high separation efficiency, our device offers a label-free separation technique and potential integration with other components, thereby serving as a promising tool for continuous cell filtration and analysis applications.

Gel-filtration analysis to detect Al-binding ligands in Eucalyptus camaldulensis

Aluminum (Al) toxicity is a major factor limiting plant growth on acid soils; inhibited root elongation is a characteristic symptom. To elucidate mechanisms of Al resistance in plants, we developed a simple method to separate Al-binding ligands from plant roots by gel-filtration chromatography. In this method, an eluent consisting of Homopipes buffer flows through a Sephadex G-25 column. Free Al ions do not pass through the column, but Al-ligand complexes can be fractionated according to differences in their molecular sizes. We applied the method to Eucalyptus camaldulensis (Myrtaceae) and successfully separated Al-binding ligands from roots. One peak of Al-ligand complex increased with Al treatment of seedlings, which can be explained by Al-induced accumulation of citrate in roots.

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.