Individual Beads Research Articles

An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries

Nucleic acids possess the unique property of being enzymatically amplifiable, and have therefore been a popular choice for the combinatorial selection of functional sequences, such as aptamers or ribozymes. However, amplification typically requires known sequence segments that serve as primer binding sites, which can be limiting for certain applications, like the screening of on-bead libraries. Here, we report a method to amplify and sequence on-bead RNA libraries that requires not more than five known nucleotides. A key element is the attachment of the starting nucleoside to the synthesis resin via the nucleobase, which leaves the 3′-OH group accessible to subsequent enzymatic manipulations. After split-and-mix synthesis of the oligonucleotide library and deprotection, a poly(A)-tail can be efficiently added to this free 3′-hydroxyl terminus by Escherichia coli poly(A) polymerase that serves as an anchored primer binding site for reverse transcription. The cDNA is joined to a DNA adapter by T4 DNA ligase. PCR amplification yielded single-band products that could be cloned and sequenced starting from individual polystyrene beads. The method described here makes the selection of functional RNAs from on-bead RNA libraries more attractive due to increased flexibility in library design, higher yields of full-length sequence on bead and robust sequence determination.

Optimizing Bead Size Improves Force Precision in Optical Traps

Optical traps are used to measure force (F) over a wide range (0.1 to 100 pN). Variation in bead radius (r) hinders comparing forces applied using different individual beads since, for small beads, trap stiffness (ktrap) varies as r3. Additionally, one does not typically calibrate ktrap for each individual bead. Rather, a prior stiffness calibration is deduced for each batch of a given bead size. This leads to systematic errors in ktrap and resulting force errors of ∼15%. Prior work has shown ktrap is maximized near 800 nm when trapping with 1064-nm light. At this maximum, variation in r should lead to minimal variation in ktrap. In this work, we show that by choosing an optimum sized bead (r = 390 nm in our trap) we reduced the standard deviation (σ) in ktrap by 2.8fold as compared to a smaller bead (250-nm) with a comparable variation in r. This improvement was validated by pulling on a DNA hairpin using a force clamp. The probability of a hairpin being open is highly sensitive to applied force - a less than 1% change in F leads to a measurable shift in the open and closed populations near F1/2 - the force at which the probability of a hairpin being open is 50%. Using this metric, the smaller beads had a σ in F1/2 of 1.2 pN while the optimum sized beads had a σ of 0.6 pN, a two-fold improvement. This improvement in precision was corroborated by the statistically indistinguishable measurement of F1/2 between both sets of data: 13.7 ± 0.4 pN and 13.6 ± 0.2 pN (mean ± SEM; N = 11). Thus, ktrap is maximized and errors in F are minimized by a simple choice of an optimum bead size.

A low-cost 2D fluorescence detection system for μm sized beads on-chip

In this paper we describe a compact fluorescence detection system for on-chip analysis of beads, comprising a low-cost optical HD-DVD pickup. The complete system consists of a fluorescence detection unit, a control unit and a microfluidic chip containing microchannels and optical markers. With these markers the laser beam of the optical pickup can be automatically focused at the centre of the microchannel. With the complete system a two-dimensional fluorescent profile across the channel width can be obtained such that there is no need for hydrodynamic or electrokinetic focusing of the particles in a specific part of the channel. Fluorescent μm sized beads suspended in medium have been detected with the system. Since on both sides of the main beam two additional laser beams at a known distance are generated, also the velocity of individual beads has been determined.

Integrated capture, transport, and magneto-mechanical resonant sensing of superparamagnetic microbeads using magnetic domain walls

An integrated platform for the capture, transport, and detection of individual superparamagnetic microbeads is described for lab-on-a-chip biomedical applications. Magnetic domain walls in magnetic tracks have previously been shown to be capable of capturing and transporting individual beads through a fluid at high speeds. Here it is shown that the strong magnetostatic interaction between a bead and a domain wall leads to a distinct magneto-mechanical resonance that reflects the susceptibility and hydrodynamic size of the trapped bead. Numerical and analytical modeling is used to quantitatively explain this resonance, and the magneto-mechanical resonant response under sinusoidal drive is experimentally characterized both optically and electrically. The observed bead resonance presents a new mechanism for microbead sensing and metrology. The dual functionality of domain walls as both bead carriers and sensors is a promising platform for the development of lab-on-a-bead technologies.

High‐throughput lectin magnetic bead array‐coupled tandem mass spectrometry for glycoprotein biomarker discovery

Alterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. High-throughput glycan profiling can now be achieved with the recent developments in MS-based techniques. To enable identification and rapid monitoring of glycosylation changes in serum proteins, we developed a semi-automated high-throughput glycoprotein biomarker discovery platform termed lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS) which includes (i) effective single-step serum glycoprotein isolation using a panel of 20 individual lectin-coated magnetic beads in microplate format, (ii) on-bead trypsin digestion, and (iii) nanoLC-MS/MS with lectin exclusion list. With use of appropriate sequence databases, LeMBA-MS can detect glycosylation changes regardless of the species. By spiking known amounts of titrated ovalbumin to a serum sample, we report nanomolar sensitivity, and linearity of response of LeMBA-MS using concanavalin A-coupled beads. Neuraminidase treatment led to reduction of binding to sialic acid-binding lectins. Interestingly, we found that desialylation caused increased binding of haptoglobin and hemopexin to mannose-specific lectins, pointing to the importance of identifying a signature of lectin-binding. High-throughput LeMBA-MS to generate glycosylation signatures will facilitate glycobiomarker discovery. LeMBA can be coupled to down-stream detection platforms for validation, making it a truly versatile platform.

Accelerated Kidney Transplant Rejection and Hypertensive Encephalopathy in a Pediatric Patient Associated With Antibodies Against Angiotensin Type 1 Receptor and HLA Class II

Accelerated Kidney Transplant Rejection and Hypertensive Encephalopathy in a Pediatric Patient Associated With Antibodies Against Angiotensin Type 1 Receptor and HLA Class II

High Throughput Screening for Antibody Responses Against H-Y Antigens and Their X-Variants in Allogeneic Hematopoeietic Stem Cell Transplantation,

Abstract 4097Allogeneic hematopoietic stem cell transplantation (alloSCT) is an effective treatment for patients with hematologic malignancies. At the Leiden University Medical Center, T-cells are depleted from the stem cell graft to reduce the incidence and severity of graft-versus-host disease (GvHD). Donor T-cells are readministered later as donor lymphocyte infusions (DLI), to preserve graft-versus-leukemia (GvL) reactivity via recognition of polymorphic peptides on recipient malignant cells.Various studies have provided evidence that not only T-cells but also antibodies play a role in the development of clinical responses (GvL and GvHD) after alloSCT. Only a limited number of polymorphic (allo) and non-polymorphic (auto) antigens, however, have been identified as targets for antibodies with potential biological relevance.We developed a high throughput Luminex bead assay for simultaneous measurement of antibodies against multiple antigens in a small sample volume. Antibody responses against antigens encoded by the Y-chromosome (H-Y antigens), which share 90–99% homology with their X-variants, have previously been reported to correlate with chronic GvHD and (maintenance of) disease remission in sex-mismatched alloSCT. We produced 3 full length H-Y antigens (RPS4Y, ZFY and DBY) as well as their respective X-variants (RPS4X, ZFX and DBX) in E.Coli as overlapping recombinant protein fragments. The protein fragments were coupled to individual Luminex bead IDs and 400 serum/plasma samples from 54 patients with chronic myeloid leukemia collected before and during treatment with alloSCT and DLI were screened for specific antibody binding to the antigen-coupled beads.Pre-existing antibodies against H-Y antigens were measured in 2/18 female and 3/36 male patients. In all of these responses ZFY was targeted, except for one female who had detectable antibodies against DBY. In 4 male patients, anti-H-Y responses were induced selectively after alloSCT (1 patient) or after subsequent DLI (3 patients). Treatment-induced antibodies against H-Y antigens were not observed in any of the female patients. Three of the 4 male patients with treatment-induced antibodies against H-Y antigens were transplanted with a female donor, out of a total of 11 female-to-male transplantations. In these patients, ZFY was targeted alone or in combination with antibodies against RPS4Y or DBY. In 1 out of 25 male-to-male transplantations, antibodies against DBY were induced after treatment with DLI.Notably, not only H-Y antigens were targets for antibodies but also their respective X-encoded homologues. Pre-existing antibodies against RPS4X, ZFX or DBX were detected in 5 patients. Of these, 1 female and 2 males also displayed antibodies against ZFY. In 2 other male recipients pre-existing antibodies against only RPS4X were measured. Furthermore, antibodies against X-variants were frequently detected selectively after alloSCT and DLI. Treatment-induced antibodies against RPS4X, ZFX or DBX were found in 3, 5 and 4 patients, respectively, and often co-developed with antibodies against the corresponding H-Y homologue. These co-developing antibodies were predominantly found in male recipients transplanted with a female donor after administration of DLI.In conclusion, we demonstrate that 3 ubiquitously expressed intracellular H-Y antigens are frequent targets for antibodies in alloSCT recipients. Treatment-induced H-Y reactive antibodies were measured most in male patients transplanted with female donors compared to other sex-matched and –mismatched settings. Antibodies against H-Y antigens often showed cross-reactivity against the respective X-encoded homologue. Our data therefore suggest that antibodies specific for intracellular H-Y and X antigens develop as a consequence of substantial lysis of patient cells during clinical responses induced after DLI. Disclosures:No relevant conflicts of interest to declare.

Guest Diffusion in Binderless High‐Performance NaX Molecular Sieves

AbstractThe pulsed field gradient (PFG) NMR technique is applied for exploring the diffusion behavior of guest molecules in binderless NaX beads in comparison with the zeolite powder employed for their production. With both probe molecules applied (water, n‐hexane), the diffusivities in the powder and the beads are found to essentially coincide as long as the diffusion path lengths are sufficiently small in comparison with the extension of the individual particles (crystallites) of the powder. With increasing diffusion path lengths, characteristic deviations become observable that can be attributed to the differences in long‐range mass transfer through the intercrystalline void volume of the bed of crystallites and within the individual beads of the binderless molecular sieve, respectively. With these studies, PFG NMR demonstrates its potentials for simultaneously recording mass transfer phenomena in both the micro‐ and macropores of commercially produced binderless molecular sieves.

Formation of an Aggregated Alginate Construct in a Tubular Perfusion System

Tissue engineering strategies are often limited by in vitro culture techniques of three dimensional scaffolds. Here we develop a method to form an aggregated cell-containing construct in vitro in a bioreactor system. Human mesenchymal stem cells (hMSCs) are cultured in individual alginate beads in a tubular perfusion system (TPS) bioreactor and then aggregated to form a single large construct. Mechanical evaluation of this construct demonstrated that aggregated alginate constructs (AACs) made from beads with 2.15 mm diameters had a Young's modulus of 85.6±15.8 kPa, a tensile strength of 3.24±0.55 kPa and a yield strength of 1.44±0.27 kPa. These mechanical properties were shown to be dependent on the bead size used to fabricate the AACs with smaller bead sizes resulting in stronger constructs. Analysis of metabolic activity revealed that hMSCs encapsulated in alginate exposed to AAC treatment sustained metabolic activity while live dead staining indicated cells remain viable. These results demonstrate the formation of AACs in the TPS bioreactor as an elegant method to create tissue engineering constructs in vitro.

Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family proteins as examples

The B-cell lymphoma-2 (Bcl-2) family contains six antiapoptotic members, each with a hydrophobic pocket in which Bcl-2 homology region 3 (BH3) helices bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins or become overexpressed under treatment. Six fusion proteins made up of glutathione-S-transferase and each of the Bcl-2 members are bound individually to six glutathione bead sets, each set being easily distinguished by its different intensity of red fluorescence. The coated bead sets are washed, combined and incubated with green fluorescent Bim-BH3 peptide and a small molecule in 10-μl wells for 1 h. The green fluorescence signal for each bead set is resolved, and selective inhibitors are expected to reduce the signal for individual bead sets. Each 384-well plate is analyzed in 12 min, measuring 200 of 2,000 beads (∼10%) of each type per well.

SU‐E‐J‐52: The Automated Detection and Scoring Algorithm for Bladder Transitional Cell Carcinoma Screening with PLZ4 Coated Beads

Purpose: One cyclic peptide named PLZ4 was identified that could selectively bind to bladder cancer cell lines and all of the 5 primary bladder cancer cells from human patients, but not to normal urothelial cells, cell mixtures from normal bladder specimens, fibroblasts, and blood cells. Beads coated with PLZ4 will have cancer cells bound on it when used for screen. It is labor intensive for human to read microscope images to make a diagnosis. A computer algorithm is developed to automate the segmentation and analysis. Methods: The computer algorithm has to complete two tasks. First, segment the image to isolate individual beads. Secondly, analysis each bead to determine whether it have cell binding. Various image processing algorithms were applied to pre‐process the image. A flood‐fill algorithm was used to isolate individual beads from background. A rejection mechanism was setup to throw away regions that are mis‐registered as beads to make the algorithm robust. The gradient magnitude of the image was used for deciding whether cell attachment exists. The regions around the edge and inside of the bead were analyzed. Scoring function was setup based on the training data. Results: The algorithm performed well in segmenting individual beads even when a lot of the beads are touching each other. Only about 1% of the beads failed to get detected. The algorithm assigns proper confidence level for cell attachment that is generally agreed by human. The result is improvable when more training data is provided. Conclusions: The automated detection and scoring algorithm for bladder transitional cell carcinoma screening with PLZ4 coated beads is promising for automatic imaging detection during diagnosis and follow‐up/surveillance of noninvasive and advanced bladder cancer.

Electron tunnelling in metallic beads systems and slow electric relaxation

We study numerically and theoretically the electrical behavior of individual steel beads and the contact between 2 adjacent beads within a granular system. Due to the thin insulating oxide layer present at the surface of the beads, the coupling between neighboring beads is likely to occur through electron tunneling. The quantum tunneling dominates at low current where it generates a high resistance in the upwards characteristic. Retaining only fundamental ingredients such as modeling the tunneling contact as a parallel RC circuit, we show how our model agrees qualitatively and quantitatively with experimental results as the observed hysteretic features and the slow relaxation of the electrical properties of a large assembly of beads.

Mechanically enforced bond dissociation reports synergistic influence of Mn2+ and Mg2+ on the interaction between integrin α7β1 and invasin

Integrins require the divalent ions magnesium and manganese for ligand recognition. Here we mechanically enforced bond dissociation to explore the influence of these ions on the mechanical strength of the specific bond between α(7) β(1) integrin and its pathologically relevant ligand invasin. Upon addition of these cations to the measurement buffer, we observe a pronounced increase in the force necessary to separate integrin and invasin coated beads. Both ions were found to work synergistically. With free invasin in the measurement buffer we furthermore observe that competitive blocking of binding sites overrides the increase in binding strength of individual beads. We show that this is due to a very strong dependence of bond affinity on divalent ions. Our study illustrates the importance of divalent ions for the regulation of force transmission by integrin ligand bonds on the molecular level.

Experimental study of dielectrophoresis and liquid dielectrophoresis mechanisms for particle capture in a droplet

This work presents a microfluidic system that can transport, concentrate, and capture particles in a controllable droplet. Dielectrophoresis (DEP), a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a non-uniform electric field, is used to manipulate particles. Liquid dielectrophoresis (LDEP), a phenomenon in which a liquid moves toward regions of high electric field strength under a non-uniform electric field, is used to manipulate the fluid. In this study, a mechanism of droplet creation presented in a previous work that uses DEP and LDEP is improved. A driving electrode with a DEP gap is used to prevent beads from getting stuck at the interface between air and liquid, which is actuated with an AC signal of 200 V(pp) at a frequency of 100 kHz. DEP theory is used to calculate the DEP force in the liquid, and LDEP theory is used to analyze the influence of the DEP gap. The increment of the actuation voltage due to the electrode with a DEP gap is calculated. A set of microwell electrodes is used to capture a bead using DEP force, which is actuated with an AC signal of 20 V(pp) at a frequency of 5 MHz. A simulation is carried out to investigate the dimensions of the DEP gap and microwell electrodes. Experiments are performed to demonstrate the creation of a 100-nL droplet and the capture of individual 10-μm polystyrene latex beads in the droplet.

Proppants: Where in the World

With the rise in hydraulic fracturing over the past decade has come a steep climb in proppant demand. Global supplies are currently tight. The number of proppant suppliers worldwide has increased since 2000 from a handful to well over 50 sand, ceramic proppant, and resin-coat producers. The estimated amount of proppant used has grown tenfold since 2000, when little more than 3 billion lbs was supplied, according to research by PropTester and Kelrik. Circumstances appear favorable for those seeking to enter the market. However, gearing up to take advantage of these propitious conditions represents a daunting task, requiring coordination of complex logistics, material resources, and processing knowledge, as well as a substantial capital investment in processing and material handling facilities. The price proppant manufacturers can charge must be figured in to the overall cost of producing a well, which in the Bakken, for example, can be approximately USD 6 million, with proppant representing about 5% of well costs. Since a majority of proppant is currently used in fracturing unconventional gas wells, depressed natural gas prices tend to keep a cap on the prices proppant suppliers can demand. However, as hydraulically fractured wells proliferate, a growing body of data is continually being generated to aid in the creation of targeted fracture stimulation plans, with more highly engineered stages—boosting the demand for greater volumes of proppant—being performed per well to maximize production. For example, a typical Barnett Shale well would in the early 2000s have consumed approximately 300,000 lbs of proppant. The longer horizontal wells drilled today, produced with often 20 or more stages, might consume 3 to 5 million lbs. Still, proppant supplier margins are relatively slim, requiring the steady, high-quality production and distribution of large volumes to sustain a profitable enterprise. Proppants: Earth to Earth The journey of an individual grain or bead of proppant is a circular one, making its way from its origins as a substance mined from the Earth to its final destination deep within the Earth in the far reaches of the filigree-like or dendritically shaped fractures emanating from the long horizontal borehole. It is the only substance operators want to remain downhole following a successful hydraulic fracturing treatment and performs the vital function of propping open the fractured formation to promote the economic flow—or conductivity—of hydrocarbons in the ensuing months and years over the well’s productive life. The primary order of business for a proppant facility, therefore, is choosing the optimum location—preferably one close to both raw material supply and end-user. The immediate proximity of railcar and possibly barge transport is critical, as well as a good road system and the availability of a reliable trucking fleet.

Development of a Multiplex Bead-Based Assay for Detection of Hepatitis C Virus

Hepatitis C virus (HCV) infection is a major burden to public health worldwide, affecting approximately 3% of the human population. Although HCV detection is currently based on reliable tests, the field of medical diagnostics has a growing need for inexpensive, accurate, and quick high-throughput assays. By using the recombinant HCV antigens NS3, NS4, NS5, and Combined, we describe a new bead-based multiplex test capable of detecting HCV infection in human serum samples. The first analysis, made in a singleplex format, showed that each antigen coupled to an individual bead set presented high-level responses for anti-HCV-positive reference serum pools and lower-level responses for the HCV-negative pools. Our next approach was to determine the sensitivity and specificity of each antigen by testing 93 HCV-positive and 93 HCV-negative sera. When assayed in the singleplex format, the NS3, NS4, and NS5 antigens presented lower sensitivity values (50.5%, 51.6%, and 55.9%, respectively) than did the Combined antigen, which presented a sensitivity of 93.5%. All antigens presented 100% specificity. These antigens were then multiplexed in a 4-plex assay, which resulted in increased sensitivity and specificity values, performing with 100% sensitivity and 100% specificity. The positive and negative predictive values for the 4-plex assay were 100%. Although preliminary, this 4-plex assay showed robust results that, aligned with its small-sample-volume requirements and also its cost- and time-effectiveness, make it a reasonable alternative to tests currently used for HCV screening of potentially infected individuals.

Deposition of anionic conjugated poly(phenylenevinylene) onto silica nanoparticles via electrostatic interactions — Assembly and single-particle spectroscopy

Fluorescent nanoparticles were prepared via adsorption of the conjugated polyelectrolyte poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV) onto 50 and 100 nm aminosilane functionalized silica beads. The particles were investigated via ensemble and single-molecule or -particle spectroscopy techniques to quantify the effect of the silica bead core on the exciton migration efficiency within the polymer. Ensemble emission spectra and ensemble fluorescence quenching studies with methyl viologen are consistent with good exciton migration along the polymer in the polymer-coated bead. The silica nanobead scaffolding preserves the sensitivity of the free polymer and provides a controllable architecture that minimizes nonspecific interactions. Single-particle spectroscopy studies were conducted on particles immobilized onto the positively charged surface of glass cover slips. Particle immobilization enabled us to monitor the effect of oxygen scavenger solutions on individual particles by changing the surrounding solution. The intensity–time trajectories of individual beads provide a mechanism of signal transduction with potential applications in multiplexing studies. Hundreds of individual beads can be imaged in a rapid parallel fashion.

Weld bead modeling and process optimization in Hybrid Layered Manufacturing

Hybrid Layered Manufacturing is a Rapid Manufacturing process in which the metallic object is built in layers using weld deposition. Each layer built through overlapping beads is face milled to remove the scales and scallops and ensure Z-accuracy. The formations of single beads and overlapping multiple beads are modeled in this paper. While the individual bead’s geometry is influenced by the size of the filler wire and the speeds of the wire and torch, the step over increment between the consecutive beads additionally comes into the picture for the multiple bead deposition. These models were validated experimentally. They are useful not only to predict the bead’s shape but also to optimize the three process parameters.

Diblock copolymer membranes investigated by single-particle tracking

We report a study on particle diffusion in membranes formed from polystyrene-block-poly(2-(dimethylamino)ethyl methacrylate) (PS-b-PDMAEMA) diblock copolymers. The membranes were investigated by scanning electron microscopy and by single-particle tracking employing carboxy-functionalized polystyrene beads loaded with a fluorophore as spectroscopic probes. From the diffusion trajectories we extracted the domain size distribution of the membranes and the local diffusion coefficient of the beads as a function of the size of the beads. The single-particle tracking data revealed that the effective domain sizes of the membranes are reduced with respect to the domain sizes obtained from scanning electron microscopy, reflecting the confined diffusion of the probe particles due to interactions with the domain walls. This is corroborated by a clear correlation between the diffusion coefficient of an individual polystyrene bead and the size of the actual domain to which it is confined.