Centrifuge Force Research Articles

Modified Pluronic F127 Surface for Bioconjugation and Blocking Nonspecific Adsorption of Microspheres and Biomacromolecules

Many experiments and applications require the chemical coupling of target molecules to surfaces, during which the elimination of nonspecific interactions presents a difficult challenge. We report on a technologically accessible surface passivation and chemical conjugation method based on an NHS end-labeled F127 Pluronic block copolymer (F127-NHS). To quantify interactions between the F127-NHS surface and magnetic microspheres, we developed a simple assay: the microsphere adhesion by gravity, inversion, then counting, or "MAGIC" assay. To improve blocking of microspheres while maintaining the ability to chemically couple additional molecules, we implemented a pH-dependent two-step chemical modification process for amine microspheres. This process achieves an extremely high level of blocking nonspecific interactions (less than 2% nonspecific adhesion) for a variety of microsphere surface charges and chemical functionalities. We also demonstrate the ability to specifically tether magnetic microspheres to an F127-NHS surface, using single DNA molecules. Using the DNA microspheres, we establish the applicability of the surface for force spectroscopy (stable with an applied load >30 pN) via the massively parallel technique of centrifuge force microscopy. Finally, we demonstrate that the surface can be used in fluorescence studies with a fluorogenic peptide cleavage assay, with high levels of blocking achieved for both the fluorogenic peptide and trypsin. These results suggest applications including, but not limited to, single-molecule force spectroscopy and fluorescence, biosensors, medical implants, and anti-biofouling, which could make use of the F127-NHS surface.

Single-Molecule Assay for Proteolytic Susceptibility: Force-Induced Destabilization of Collagen's Triple Helix

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless Mini-Radio Centrifuge Force Microscope (MR.CFM) to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules were used to determine how the triple helix responds to force. As the primary load- and tension-bearing protein in vertebrates, the physical properties of collagen are of significant biomedical interest. How collagen's triple helix responds to applied force is controversial, with different studies inferring incompatible outcomes: overwinding, unwinding, shearing, or maintaining its zero-force structure. Because proteolytic cleavage requires a locally unwound triple helix, our experiments reveal how local collagen structure changes in response to applied force. Our first results show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix. The generality of this result will be discussed in the context of collagen's sequence heterogeneity.

Single-Molecule Assay for Proteolytic Susceptibility: Force-Induced Collagen Destabilization

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.

Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy.

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as high cost, low throughput, technical difficulty, and strict infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope (CFM) adapted to a commercial centrifuge enables use by nonspecialists, and integration with DNA nanoswitches facilitates both reliable measurements and repeated molecular interrogation. Here, we provide detailed protocols for constructing the CFM, creating DNA nanoswitch samples, and carrying out single-molecule force measurements.

Nanoemulsion for oil-contaminated oil-based drill cuttings removel in lab

This paper demonstrates a new nanoemulsion NA-R, is an oil-in-water emulsion. It shows the natural distribution of <100 nm particle size. NA-R is able to provide ultralow surface tension of 33 mN/m and ultralow interfacial tension of 1.35 mN/m. When using it to remove the oil from the cuttings, it is able to reduce the contact angle and capillary force on the cuttings particle surface, subsequently, allowed water to penetrate and wet the particle surface and accessible pores. Once the particles surfaces are water-wet, oil will instantly repel from it and easily segregate through centrifuge force. It was found that the oil removal rate in the cuttings after treatment with nanoemulsion cleaning process was able to reach 95.7%.

Manual centrifuge system: Bearing-based hand spinner made with 3-D printer.

Compact disk (CD) Microfluidic platforms are being studied for medical applications such as blood tests. However, its size is bulky and electricity is needed to realize centrifuge force. In this paper, bearing-based hand spinner is designed using three-dimensional printer. This spinner does not need electricity and keeps rotating direction unlike paperfuge while it is spinning. The properties of spinner vary depending on bearing type which is positioned at the center. The type of weighting area also affects change in RPM over time. When a separation experiment is implemented, separating mixture into red ink and oil and whole blood into red blood cell and plasma are achieved properly with ceramic ball bearing.

Enhancing the Detection of Dysmorphic Red Blood Cells and Renal Tubular Epithelial Cells with a Modified Urinalysis Protocol

Urinary sediment is used to evaluate patients with possible urinary tract diseases. Currently, numerous protocols are applied to detect dysmorphic red blood cells (RBCs) and renal tubular epithelial cells (RTECs) in urinary sediment. However, distinct protocols are used by nephrologists and medical technologists for specimen concentration and observation, which leads to major discrepancies in the differential counts of formed elements such as dysmorphic RBCs and RTECs and might interfere with an accurate clinical diagnosis. To resolve these problems, we first tested a modified urinalysis protocol with an increased relative centrifuge force and concentration factor in 20 biopsy-confirmed glomerulonephritis patients with haematuria. We successfully improved the recovery ratio of dysmorphic RBCs in clinical specimens from 34.7% to 42.0% (P < 0.001). Furthermore, we confirmed the correlation between counts by the modified urinary protocol and Sysmex UF-1000i urinary flow cytometer (r ≥ 0.898, P < 0.001). A total of 28 types of isomorphic and dysmorphic RBCs were detected using a bright field microscope, with results comparable to those using a standard phase contrast microscope. Finally, we applied Sternheimer stain to enhance the contrast of RTECs in the urinary sediments. We concluded that this modified urinalysis protocol significantly enhanced the quality of urinalysis.

Two-dimensional spin coating with a vertical centrifugal force and the effect of artificial gravity on surface leveling

This study focuses on an innovative method for spin coating called the two-dimensional (2D) spin coating method. Using a centrifugal force applied by a rotary machine perpendicular to the wafer surface body, a vertical centrifuge force (VCF) was generated. The VCF allowed controllable artificial gravity acceleration to be generated and caused the coating to face this elevated gravity acceleration to adjust and normalize the high and low surface tension stresses. Previous surface leveling mathematics were analyzed and modified. The modified calculations indicate that the effect of additional gravity exerted on the liquid’s surface can reduce the amplitude of surface leveling. To experimentally investigate this phenomenon, a 2D spin coater was designed and manufactured. Higher artificial gravity overcame some common coating defects, such as cloudiness, edge beading, inner layer bubbling, and unsmooth surface leveling. Photoresist (AZP4620) was used as the coating material. The surface roughness was analyzed by atomic force microscopy (AFM) and the layer properties were also imaged by scanning electron microscopy (SEM). The AFM results (average and root-mean-square roughness) indicated a decrease in surface leveling amplitude by increasing the VCF. SEM images showed condensed layers without any porosity or rupture. The experimental results agreed with the simulations and calculated values.

A wireless centrifuge force microscope (CFM) enables multiplexed single-molecule experiments in a commercial centrifuge

The centrifuge force microscope (CFM) was recently introduced as a platform for massively parallel single-molecule manipulation and analysis. Here we developed a low-cost and self-contained CFM module that works directly within a commercial centrifuge, greatly improving accessibility and ease of use. Our instrument incorporates research grade video microscopy, a power source, a computer, and wireless transmission capability to simultaneously monitor many individually tethered microspheres. We validated the instrument by performing single-molecule force shearing of short DNA duplexes. For a 7 bp duplex, we observed over 1000 dissociation events due to force dependent shearing from 2 pN to 12 pN with dissociation times in the range of 10-100 s. We extended the measurement to a 10 bp duplex, applying a 12 pN force clamp and directly observing single-molecule dissociation over an 85 min experiment. Our new CFM module facilitates simple and inexpensive experiments that dramatically improve access to single-molecule analysis.

Multiplexed single-molecule force spectroscopy using a centrifuge.

We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.

Multiplexed Force Spectroscopy using DNA Nanoswitch a Centrifuge

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as cost, throughput, technical difficulty, and infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope adapts to a commercial centrifuge enabling use by non-specialists, and integration of DNA nanoswitches facilitates reliable measurements. We demonstrate new features by measuring temperature and magnesium dependent DNA unzipping together with multiplexed DNA force-extension and overstretching.

Vibration-based identification of rotating blades using Rodrigues' rotation formula from a 3-D measurement

In this study, the geometrical setup of a turbine blade is tracked. A research-scale rotating turbine blade system is setup with a single 3-axes accelerometer mounted on one of the blades. The turbine system is rotated by a controlled motor. The tilt and rolling angles of the rotating blade under operating conditions are determined from the response measurement of the single accelerometer. Data acquisition is achieved using a prototype wireless sensing system. First, the Rodrigues\' rotation formula and an optimization algorithm are used to track the blade rolling angle and pitching angles of the turbine blade system. In addition, the blade flapwise natural frequency is identified by removing the rotation-related response induced by gravity and centrifuge force. To verify the result of calculations, a covariance-driven stochastic subspace identification method (SSI-COV) is applied to the vibration measurements of the blades to determine the system natural frequencies. It is thus proven that by using a single sensor and through a series of coordinate transformations and the Rodrigues\' rotation formula, the geometrical setup of the blade can be tracked and the blade flapwise vibration frequency can be determined successfully.

Development of a Wireless, Modular Centrifuge Force Microscope for Use in a Commercial Benchtop Centrifuge

A few years ago, we introduced the Centrifuge Force Microscope (CFM) as a new platform for massively parallel single-molecule manipulation. Our original prototype instrument used a microscope on a rotary stage to generate uniform centrifugal forces, which were applied to thousands of microspheres at once, hundreds of which formed single molecule tethers. Here we present a second generation CFM that is fully integrated into a commercial benchtop centrifuge, further increasing the accessibility of the method and greatly expanding the dynamic force range. We have condensed a complete video microscope to fit into a centrifuge bucket, along with battery power and an onboard computer. The result is a fully wireless “CFM module” that can be placed inside a swinging bucket, while still retaining full control of the instrument (even during centrifugation) from a wifi enabled computer. We have demonstrated the unit up to 1000g, which gives a force range of ∼70fN to 70pN using 2.8 micron microspheres. Our current device can save images at near USB 2.0 speeds with up to 5-megapixel resolution, and we are working toward improving the bandwidth further. While a number of single-molecule applications exist, one of our primary interests will be to probe the structure and function of various non-coding RNAs.

Investigating Force-Induced Structural Changes in Single Collagen Molecules

Collagen is a structural protein found in abundance throughout the body, within the extracellular matrix and connective tissues including skin, bone, and tendons. One of the functions of collagen is to maintain tissue structure in the presence of external forces. However the effect of force on the collagen's structure is not clear:1 in the presence of an external force, collagen has been proposed to have one of two opposing structural changes: either overwinding, reducing its cleavage2 or underwinding, increasing its cleavage.3 Using a high-throughput single-molecule stretching instrument, the centrifuge force microscope (CFM),4 we are developing a technique to investigate force-dependent structural changes of collagen under various loading conditions. Here, we report on our progress tethering, enzymatically cleaving and applying a tunable external force to single molecules of collagen. In addition, we present characterization of the instrument and technique accomplished with single DNA molecules.1.Chang, S.W. and Buehler M. (2014) Molecular biomechanics of collagen molecules. Mat. Today. 17, 70-76.2.Camp, R., Liles, M., Beale, J., Saeidi, N., Flynn, BP., Moore, E., Murthy, S., and Ruberti, JW. (2011) Molecular Mechanochemistry: Low Force Switch Slows Enzymatic Cleavage of Human Type I Collagen Monomer. JACS, 133, 4073-8.3.Adhikari, A., Glassey, E. and Dunn, AR. (2012) Conformation Dynamics Accompanying the Proteolytic Degradation of Trimeric Collagen I by Collagenases. JACS, 134, 13259-65.4.Halvorsen, K. and Wong, W. (2010) Massively Parallel Single-Molecule Manipulation Using Centrifugal Force. Biophys J., 98, L53-L55.

Fractionation of oats into products enriched with protein, beta-glucan, starch, or other carbohydrates

A modified wet method was developed to fractionate ground oat groats into 4 fractions enriched with beta-glucan (BG), protein, starch, and other carbohydrates (CHO), respectively. Effects of defatting oats and centrifuge force for separation were also investigated. Results show that, depending on the two factors, nutrient concentrations in the corresponding fractions ranged 28.53–44.84% (dry matter) for BG, 72.41–92.62% for protein, 79.13–81.69% for starch, and 37.47–42.16% for other CHO. Nutrient recoveries from each fraction were 39.82–51.20% for BG, 60.36–72.08% for protein, 77.04–87.25% for starch, and 24.87–29.68% for other CHO. For the protein fraction, defatting improved the protein content but did not on its recovery. For the starch fraction, the effect of defatting was just opposite, improving the starch recovery but not the starch content. Centrifuge force increased the BG content in the BG fraction when the defatted sample was used. The cumulative recovery of each component in the combined fractions was not affected by oil removal or centrifuge force. Overall, the wet method described was relatively effective in recovering the major nutrients from oats into their respective fractions, while it alleviated the problems of viscous slurry upon mixing ground oat groats with an alkaline solvent and the difficulty of slurry separation.

Methods to Recover Value-Added Coproducts from Dry Grind Processing of Grains into Fuel Ethanol

Three methods are described to fractionate condensed distillers solubles (CDS) into several new coproducts, including a protein-mineral fraction and a glycerol fraction by a chemical method; a protein fraction, an oil fraction and a glycerol-mineral fraction by a physical method; or a protein fraction, an oil fraction, a mineral fraction, and a glycerol fraction by a physicochemical method. Processing factors (ethanol concentration and centrifuge force) were also investigated. Results show that the three methods separated CDS into different fractions, with each fraction enriched with one or more of the five components (protein, oil, ash, glycerol and other carbohydrates) and thus having different targeted end uses. Furthermore, because glycerol, a hygroscopic substance, was mostly shifted to the glycerol or glycerol-mineral fraction, the other fractions had much faster moisture reduction rates than CDS upon drying in a forced air oven at 60 °C. Thus, these methods could effectively solve the dewatering problem of CDS, allowing elimination of the current industrial practice of blending distiller wet grains with CDS for drying together and production of distiller dried grains as a standalone coproduct in addition to a few new fractions.

Influence of the gravity on the discharge of a silo

We performed a series of experiments to investigate the flow of an assembly of non-cohesive spherical grains in both high and low gravity conditions (i.e. above and under the Earth’s gravity). In high gravity conditions, we studied the flow of glass beads out of a cylindrical silo and the flow of metallic beads out of a vertical Hele-Shaw rectangular silo. Both silos were loaded in one of the gondolas of the large diameter centrifuge facility (located at ESTEC) in which an apparent gravity up to 20 times the Earth’s gravity can be established. To simulate low gravity conditions, we submitted a horizontal monolayer of metallic beads to the centrifuge force of a small rotation device (located at University of Liege). The influences of both gravity and aperture size on the mass flow were analysed in these various conditions. For the three systems (cylindrical silo, the Hele-shaw silo and the monolayer of beads), we demonstrated that (i) the square root scaling of the gravity found by Beverloo is relevant and (ii) the critical aperture size below which the flow is jammed does not significantly increase with the apparent gravity. Moreover, we studied in more details the Hele-Shaw silo in high gravity because this configuration allowed to determine local properties of the flow at the level of the aperture. We measured the velocity profiles and the packing fraction profiles for the various aperture sizes and apparent high gravities. We demonstrate the existence of a slip length for the flow at the level of the aperture. This later fact seems to result from the geometrical configuration of the silo.

A new finite element for transverse vibration of rectangular thin plates under a moving mass

In this paper a new finite element which can be used in the analysis of transverse vibrations of the plates under a moving point mass is presented. In this technique, which allows for the inclusion of inertial effects of the moving mass, the load is replaced with an equivalent finite element. By means of using the relations between nodal forces and nodal deflections of 16 DOF conforming plate element with C(1) continuity, on the one hand, and shape functions, on the other hand, mass, stiffness, and damping matrices of the new finite element are determined by the transverse inertia force, Coriolis force and centrifuge force, respectively. This method was first applied on a simply supported beam so as to provide a comparison with the previous studies in the literature, and it was proved that the results were within acceptable limits. Second, it was applied on a cantilevered plate so as to determine the dynamic response of the planer entry plate of a high-speed wood-cutting machine.

Transient analysis of LP rotor from NPP 900MW turbine

Thermal stress and the contact stress for centrifuge force field during the start up and shut down is very most important for the safety of the turbine which will affect the design life of the turbine. The stress at startup and shut down is much larger than the stress at other conditions. The stress level and the fatigue life are important for safety and economy of the rotor. In this paper, the mechanical properties of the material varying with the temperature are considered. The vapor pressure and temperature at different position of the rotor and at different history are considered to calculate the film coefficient. The two dimensional thermal-mechanical coupled model is used to calculate the transient temperature field and stress field. The three dimensional contact model is used to calculate the stress field and contact stress under the centrifuge loading conditions.

Influence of a reduced gravity on the volume fraction of a monolayer of spherical grains

Centrifuge force is used to study granular materials in low gravity conditions. We consider a monolayer of noncohesive spherical grains placed on a plate. Reduced gravity conditions can be simulated in the plane by tilting or by rotating the plate. We compare both approaches experimentally. The volume fraction is found to increase with the apparent gravity and saturates. A model based on the exponential distribution of the Voronoi cell areas has been built and is in excellent agreement with the experimental data by extrapolating the fits of the data. Moreover, numerical simulations exhibit that more arches can be maintained at low apparent gravities than at high.