Capture Yield Research Articles

Plasminogen-Based Capture Combined with Amplification Technology for the Detection of PrPTSE in the Pre-Clinical Phase of Infection

BackgroundVariant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrPTSE) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrPTSE concentrations in the femtomolar range.Methodology/Principal FindingsWe have developed a three-step assay that firstly captures PrPTSE from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrPTSE detection by western blot. We achieved a PrPTSE capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrPTSE in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrPTSE in human plasma spiked with a 10−8 dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrPTSE in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.Conclusion/SignificanceWe have developed a sensitive and specific amplification assay allowing the detection of PrPTSE in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrPTSE in blood of patients displaying positivity in large scale screening tests.

High throughput capture of circulating tumor cells using an integrated microfluidic system

In this work, we introduced an integrated microfluidic system for fast and efficient circulating tumor cell (CTC) isolation and capture. In this microfluidic platform, a combination of microfluidic deterministic lateral displacement array and affinity-based cell capture architecture, allows for the high efficiency cancer cell enrichment and continuous high throughput and purity cancer cell capture. Using this device to isolate breast cancer cells from spiked blood samples, we achieved an enrichment factor of 1500×, and a high processing throughput of 9.6mL/min with 90% capture yield and more than 50% capture purity at cell concentration 102cells/mL. This integrated platform offers a promising approach for CTC capture with high recovery rates, purity and stability, and exhibits potential capability in cancer cell culture and drug screening.

A microbead-incorporated centrifugal sample pretreatment microdevice

In this study, we present a novel bead-incorporated centrifugal sample pretreatment microdevice to purify influenza A H3N2 viral RNA. Simple revolution per minute (RPM) control can lead to RNA capture on a bead-bed, and the sequential loading of a washing solution and an elution solution. Tetraethoxy orthosilicate (TEOS)-treated glass microbeads were utilized as a capture matrix. The sample pretreatment microdevice consists of four reservoirs for storing an RNA sample, a washing solution, an elution solution, and a collected sample, and they were merged at the microbead-bed microchannel. The washing solution reservoir and the elution solution reservoir were connected to the bead-bed microchannel through a capillary valve and a siphon channel, respectively. An RNA sample (a lysed influenza A H3N2 virus), a washing solution (70% ethanol) and an elution solution (water or a reverse transcription-polymerase chain reaction (RT-PCR) cocktail) were loaded into the designated reservoirs, and they were successively transported to the bead-bed by RPM control owing to the optimized channel design. Purified RNAs could be obtained in 440 s. Then, a target H3 gene was amplified by an off-chip based real-time RT-PCR to evaluate the capture efficiency of RNA on our proposed microdevice. 81% of RNAs were successfully captured and purified. Interestingly, the use of the RT-PCR cocktail itself as an elution solution resulted in a 76% capture yield. Furthermore, we successfully performed RNA purification from the clinical nasopharyngeal swabs to identify the subtype of the influenza A virus. This platform provides high potential for the direct integration of the sample pretreatment microdevice into the downstream micro-PCR unit to create a total genetic analysis microsystem.

Validation of capture yield calculations in the Resolved Resonance Energy Range with CONRAD code

This paper deals with the validation of the multiple scattering corrections developed in the CONRAD code for the capture yield calculations in the Resolved Reso- nance energy Range (RRR). In order to calculate the capture yields, analytic and stochas- tic calculation schemes implemented in CONRAD are described and compared with the analysis code SAMMY/SAMSMC. The results are in excellent agreement for a variety of samples. We concentrate the discussion here on 238 U, 197 Au and 55 Mn.

Neutron induced capture and fission discrimination using calorimetric shape decomposition

The neutron capture and fission cross-sections of 233U have been measured at the neutron time-of-flight facility n_TOF at CERN in the energy range from 1eV to 1keV using a high performance 4π BaF2 Total Absorption Calorimeter (TAC) as a detection device. In order to separate the contributions of neutron capture and neutron induced fission in the TAC, a methodology called Calorimetric Shape Decomposition (CSD) was developed. The CSD methodology is based on the study of the TAC's energy response for all competing reactions, allowing to discriminate between γ s originating from neutron induced fission and those from neutron capture reactions without the need for fission tagging or any additional detection system. In this article, the concept behind the CSD is explained in detail together with the necessary analysis to obtain the TAC's response to neutron capture and neutron induced fission. The discrimination between capture and fission contributions is shown for several neutron energies. A comparison between the 233U neutron capture and fission yield extraction with ENDF/B-VII v1. library data is also provided.

Cell rolling and adhesion on surfaces in shear flow. A model for an antibody-based microfluidic screening system

Graphical abstractDisplay Omitted Highlights? We use a microfluidic chamber to investigate the cell rolling. ? We examine changes of cell behavior according to the hydrodynamic parameters. ? We schematize the cell behavior from firm adhesion to free rolling regime with the shear stress. ? We model the cell rolling accounting the adhesion force represented by a linear spring. ? The good fitting between numerical and experimental describes the cell behavior. Here we present a model and an experimental investigation to study cell bound to the floor of a microfluidic system and the flow induced detachment. The experimental investigation has been performed by a microfluidic assay biofunctionalized with specific antibodies. The upregulation of the cell membrane density of specific antigens has been exploited to detect and concentrate cells using hydrodynamic forces. The numerical model explored the role that the hydrodynamic forces have on adhesion-detachment of cell to the biofunctionalized substrate. To account the adhesion force, the cell receptor-surface ligand interaction has been represented by a linear spring exerting adhesive force on the target cell. The experimental investigation with the W6/32, an antibody that binds specifically to MHC class I molecule and which has an important role in the recognition of the tumor cells from the immune system, has been simulated by the numerical model with a constant spring Ks=7.5i?10-8N/s. The velocity of cells and of the fluid and the experimental capture yield have been compared. The existence of three different regimes of cell behavior has been shown, moving from firm adhesion to free rolling. Up to 30µl/s the cells experience the adherent rolling, whilst at higher flow rate the cells start to move with the fluid in a regime of free rolling. The model provides physical insight, explaining apparently counterintuitive features of the prototype assay data.

Measurement of resolved resonances of232Th(n,γ) at the n_TOF facility at CERN

The yield of the neutron capture reaction 232Th(n,γ) has been measured at the neutron time-of-flight facility n_TOF at CERN in the energy range from 1 eV to 1 MeV. The reduction of the acquired data to the capture yield for resolved resonances from 1 eV to 4 keV is described and compared to a recent evaluated data set. The resonance parameters were used to assign an orbital momentum to each resonance. A missing level estimator was used to extract the s-wave level spacing of D0=17.2±0.9 eV.9 MoreReceived 13 February 2012Publisher error corrected 20 June 2012DOI:https://doi.org/10.1103/PhysRevC.85.064601©2012 American Physical Society

Solution-based targeted genomic enrichment for precious DNA samples

BackgroundSolution-based targeted genomic enrichment (TGE) protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1) modifying or eliminating time consuming steps; 2) increasing yield to reduce input DNA and excessive PCR cycling; and 3) enhancing reproducible.ResultsWe developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company.ConclusionsThis solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

Measurement and resonance analysis of the237Np neutron capture cross section

The neutron capture cross section of 237Np was measured between 0.7 and 500 eV at the CERN n_TOF facility using the 4π BaF2 Total Absorption Calorimeter. The experimental capture yield was extracted minimizing all the systematic uncertainties and was analyzed together with the most reliable transmission data available using the sammy code. The result is a complete set of individual as well as average resonance parameters [D0=0.56(2) eV, 〈Γγ〉=40.9(18) meV, 104S0=0.98(6), R′=9.8(6) fm]. The capture cross section obtained in this work is in overall agreement with the evaluations and the data of Weston and Todd [Nucl. Sci. Eng. 79, 184 (1981)], thus showing sizable differences with respect to previous data from Scherbakov et al. [J. Nucl. Sci. Technol. 42, 135 (2005)] and large discrepancies with data Kobayashi et al. [J. Nucl. Sci. Technol. 39, 111 (2002)]. The results indicate that a new evaluation combining the present capture data with reliable transmission data would allow reaching an accuracy better than 4%, in line with the uncertainty requirements of the nuclear data community for the design and operation of current and future nuclear devices.8 MoreReceived 28 November 2011DOI:https://doi.org/10.1103/PhysRevC.85.044616©2012 American Physical Society

Automation Highlights from the Literature

Automation Highlights from the Literature

A rotary microsystem for simple, rapid and automatic RNA purification

In this work, we demonstrate a novel rotary microsystem for simple, rapid and automatic influenza viral RNA purification. The microdevice consists of a silica sol-gel matrix for RNA capture, and three reservoirs for a RNA sample (R(S)), a washing solution (R(W)) and an elution buffer (R(E)) that were connected with different dimensional microfluidic channels (120 μm for R(S), 40 μm for R(W), and 20 μm for R(E)). The hydrophobic property of PDMS and the narrow microchannel served as a passive capillary microvalve, and the loading of the solutions were controlled by centrifugal force. 5 μL of a lysate sample of influenza A H1N1 virus, a washing solution and an elution buffer were injected in each designated reservoir, and the virus sample, the washing solution, and the elution buffer were sequentially loaded into the sol-gel chamber at 1600, 2000, and 2500 RPM, enabling the viral RNA to be captured in the sol-gel solid phase, purified, and eluted in 5 min. The RNA capture yield was measured as ~80%, and the H1 and M gene were successfully amplified from the recovered purified H1N1 viral RNA by reverse-transcriptase PCR. Such a novel rotary sample preparation system eliminates any complicated hardware and human intervention, and performs the RNA extraction with high speed and high fidelity.

Parallel single‐cell analysis microfluidic platform

We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed. First, we report single-cell trapping in a fast (2-5 min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content. It is validated here using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content.

Modeling Filtration of Platelet-Rich Plasma in Fibrous Filters

Removing leukocytes (white cells) from blood products such as platelet-rich plasma (PRP) prevents serious problems for the PRP recipients. We present a model to study selective separation of leukocytes from a dilute suspension of leukocytes and platelets (PRP) using fibrous filters. A selective PRP filter permits platelets to pass but bars the way for leukocytes. In PRP filters, fine synthetic fibers are packed randomly and interstices are larger than cells. Interactive forces between the suspended cells and fibers determine cell capture yield, i.e., deep-bed filtration process. Our model is based on a hierarchical set of differential equations corresponding to porescale and macroscale. At the porescale, we model movement and interaction of cells with fibers. A single cell entering the interstitial space encounters drag and surface forces. We define a unit bed element (UBE) composed of a fiber and interstitial space, where fiber diameter corrected for the size of the cells. We add measured surface forces between cells and fibers (electrostatic, van der Waals, fluid expression resistance, and biological responses) to the cell velocity equation. We then transform pore scale events into macroscale continuum by imposing periodic boundary conditions for contiguous UBEs and applying macrotransport theory. At macroscale, two independent coefficients: mean cell velocity vector U* and mean cell capture yield constant K*characterize cell filtration efficiency. Our model showed reasonable match with experimental data. Model results showed that diameter of fibers, injection velocity, bed porosity, and bed thickness control selective capture of leukocytes. Our model showed that it is merely impossible to filter out all leukocytes without capturing platelets. Based on our model, an optimized filter can capture 90% of leukocytes and pass through 45% of platelets.

Effect of Surface Nanotopography on Immunoaffinity Cell Capture in Microfluidic Devices

Immunoaffinity microfluidic devices have recently become a popular choice to isolate specific cells for many applications. To increase cell capture efficiency, several groups have employed capture beds with nanotopography. However, no systematic study has been performed to quantitatively correlate surface nanopatterns with immunoaffinity cell immobilization. In this work, we controlled substrate topography by depositing close-packed arrays of silica nanobeads with uniform diameters ranging from 100 to 1150 nm onto flat glass. These surfaces were functionalized with a specific antibody and assembled as the base in microfluidic channels, which were then used to capture CD4+ T cells under continuous flow. It is observed that capture efficiency generally increases with nanoparticle size under low flow rate. At higher flow rates, cell capture efficiency becomes increasingly complex; it initially increases with the bead size then gradually decreases. Surprisingly, capture yield plummets atop depositions of some particle diameters. These dips likely stem from dynamic interactions between nanostructures on the substrate and cell membrane as indicated by roughness-insensitive cell capture after glutaraldehyde fixing. This systematic study of surface nanotopography and cell capture efficiency will help optimize the physical properties of microfluidic capture beds for cell isolation from biological fluids.

Differential magnetic catch and release: experimental parameters for controlled separation of magnetic nanoparticles

Differential magnetic catch and release (DMCR) has been used as a method for the purification and separation of magnetic nanoparticles. DMCR separates nanoparticles in the mobile phase by magnetic trapping of magnetic nanoparticles against the wall of an open tubular capillary wrapped between two narrowly spaced electromagnetic poles. Using Au and CoFe(2)O(4) nanoparticles as model systems, the loading capacity of the 250 μm diameter capillary is determined to be ∼130 μg, and is scalable to higher quantities with larger bore capillary. Peak resolution in DMCR is externally controlled by selection of the release time (R(t)) at which the magnetic flux density is removed, however, longer capture times are shown to reduce the capture yield. In addition, the magnetic nanoparticle capture yields are observed to depend on the nanoparticle diameter, mobile phase viscosity and velocity, and applied magnetic flux. Using these optimized parameters, three samples of CoFe(2)O(4) nanoparticles whose diameters are different by less than 10 nm are separated with excellent resolution and capture yield, demonstrating the capability of DMCR for separation and purification of magnetic nanoparticles.

Herceptin functionalized microfluidic polydimethylsiloxane devices for the capture of human epidermal growth factor receptor 2 positive circulating breast cancer cells.

Building on recent breakthroughs in the field of microfluidic-based capture of rare cancer cells circulating in the blood, the present article reports on the use of Herceptin functionalized PDMS devices designed to efficiently capture from blood cancer cells, overexpressing the tyrosine kinase human epidermal growth factor receptor (HER2). The identification of patients overexpressing HER2 is critical as it typically associates with an aggressive disease course in breast cancer and poor prognosis. Importantly, HER2 positive patients have been found to significantly benefit from Herceptin (Trastuzumab), a humanized monoclonal antibody (MAb) against HER2. Disposable PDMS devices prepared using standard soft lithography were functionalized by the plasma polymerization of an epoxy-containing monomer. The epoxy-rich thin film (AGEpp) thus created could be conjugated with Herceptin either directly or through a polyethylene glycol interlayer. The properties and reactivity toward the monoclonal antibody conjugation of these coatings were determined using x-ray photoelectron spectroscopy; direct conjugation provided a good compromise in reactivity and resistance to biologically nonspecific fouling and was selected. Using the breast cancer cell line SK-BR-3 as a model for cells overexpressing HER2, the immunocapture efficacy of the Herceptin functionalized PDMS was demonstrated in model studies. Validation studies confirmed the ability of the device to efficiently capture (∼80% capture yield) HER2 positive cells from full blood.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Regulation of tillering in sorghum: environmental effects.

Tillering has a significant effect on canopy development and, hence, on resource capture, crop growth and grain yield in sorghum. However, the physiological basis of tillering and its regulation by environmental effects are not fully understood. The objective of this study was to understand and quantify the environmental effects on tillering in sorghum using a carbohydrate supply-demand framework. A series of five experiments with a wide range of radiation and temperature conditions was conducted and details of the tillering responses of a single representative hybrid were monitored. The concept of internal plant competition for carbohydrate was developed for analysis of these responses. Tiller appearance was highly synchronized with main shoot leaf appearance, with a consistent hierarchy for tillering across environments. The main environmental effect was on the frequency of tiller appearance, in particular of the lower-rank tillers. This explained some of the observed environmental differences in the onset of tillering. A generalized index of internal plant competition, which took account of plant assimilate supply and demand (S/D(index)) during the critical period for tillering, explained most of the variation in maximum tiller number observed across the five experiments. This result was consistent with the hypothesis that internal plant competition for assimilates regulates tillering in sorghum. Hence, the framework outlined has a predictive value that could provide the basis for dynamic simulation of tillering in crop growth models.

Delineation of management zones using mobile measurements of soil apparent electrical conductivity and multivariate geostatistical techniques

Site-specific management promotes the identification and management of areas within the field, which represent subfield regions with homogeneous characteristics (management zones). However, determination of subfield areas is difficult because of the complex combination of factors which could affect crop yield. One possibility to capture yield variability is the use of soil physical properties to define the management zones as they are related to plant available water. With the aim of characterizing the spatial variability of the main soil physical variables and using this information to determine potential management zones, soil samples were taken from 70 locations in a 33-ha field in Badajoz, southwestern Spain. Firstly, accurate spatial distribution maps of the soil attributes were generated by using regression kriging as the most suitable algorithm in which exhaustive secondary information on soil apparent electrical conductivity (ECa) was incorporated. ECa measurements were carried out with a Veris 3100 operating in both shallow (0–30 cm), ECs, and deep (0–90 cm), ECd, mode. Clay, coarse sand and fine sand were the soil physical properties which exhibited higher correlation with ECa (positively correlated with the finer texture component, clay, and negatively correlated with the coarser ones, coarse and fine sands), particularly with ECs. Thus, this was the secondary variable used to obtain the kriged maps. Later, principal component analysis and fuzzy cluster classification were performed to delineate management zones, resulting in two subfields to be managed separately. This number of subfields was determined using the fuzzy performance index and normalized classification entropy as the way to optimize the classification algorithm.

Measurement of keV-neutron capture cross-sections and capture γ-ray spectra of 56Fe and 57Fe

The neutron capture cross-sections and the capture γ-ray spectra of 56Fe and 57Fe have been measured in the neutron energy range from 10 to 90 keV. Pulsed keV-neutrons were produced from the 7Li( p, n) 7Be reaction by bombarding a lithium target with a 1.5-ns bunched proton beam from a 3 MV Pelletron accelerator. The incident neutron spectrum on the capture sample was measured using a time-of-flight method with a 6Li-glass detector. The capture γ-rays emitted from an iron or standard gold sample were detected with a large anti-Compton NaI(Tl) spectrometer. The capture yield of the iron or gold sample was obtained by applying a pulse-height weighting technique to the corresponding capture γ-ray pulse-height spectrum. The capture cross-sections of 56,57Fe were derived with errors less than 5% using the standard capture cross-sections of 197Au. The capture γ-ray spectra were obtained by unfolding the observed capture γ-ray pulse-height spectra. The present results for the capture cross-sections were compared with the previous measurements and the evaluated values of ENDF/B-VII.0 and JENDL-3.3. The Maxwellian-averaged capture cross-sections of 56Fe and 57Fe at 30 keV are derived as 12.22 ± 2.06 mb and 44.48 ± 7.56 mb, respectively.