Circular Probe Research Articles

Biphenyl dioxolanes as circular dichroism probes for the assignment of absolute configuration to aliphatic diols: Extending the scope to anti 1,n‐diols and cyclic syn 1,2‐diols

We describe herein the use of a flexible biphenyl moiety as efficient chirality probe in the assignment of the absolute configuration (AC) of aliphatic, non-chromophoric diols. The diols are transformed in the corresponding biphenyl dioxolanes in which the biphenyl system has either a P or M torsion depending on the chirality of the diol. As the correlation between biphenyl torsion and diol AC has been established and the sense of torsion is revealed by the sign of the biphenyl A band at 250 nm in the CD spectrum of the dioxolane, then the diols AC can be assigned simply looking at the CD spectra of these derivatives. This approach proved to be general, straightforward, and reliable for anti 1,2- 1,3-, and 1,4-diols bearing both one and two stereogenic centers and for cyclic syn 1,2-diols.

A Circular Dichroism Probe for L‐Cysteine Based on the Self‐Assembly of Chiral Complex Nanoparticles

A Circular Dichroism Probe for <scp>L</scp>‐Cysteine Based on the Self‐Assembly of Chiral Complex Nanoparticles

Determination of the Optimal Ball Grinding Parameters for the Bio-Ceramics ZrO&lt;sub&gt;2&lt;/sub&gt; on a PC-Based Multi-Axis CNC Engraving Machine

The objective of this study is to determine the optimal ball grinding parameters of the bio-ceramics ZrO2 by executing the Taguchi’s L18 orthogonal array experiment, analysis of variation (ANOVA), and the full factorial experiment on a PC-based multi-axis CNC engraving machine. The manufacture of a ZrO2 coping using the optimal ball grinding parameters has also been studied based on a tooth plaster model via reverse engineering technique. The 3D data points of a tooth plaster model were digitized by the developed on-machine measurement system on a PC-based CNC multi-axis engraving machine by integrating a circular triangulation laser probe with the machine and a PC. A CAD model of a coping could then be constructed by Pro/Engineer software, based on the digitized 3D data points. With the help of the PowerMill CAM software, the machining path of a coping has been simulated, and the correspondent NC codes have been generated to fabricate the ZrO2 coping using the determined optimal ball grinding parameters, on the CNC multi-axis engraving machine. The average surface roughness of Ra=0.49 um for the ground ZrO2 ceramics coping could be obtained by utilizing the optimal flat surface grinding parameters.

Rolling Circle Amplification for Signal Enhancement in Ovalbumin Detection

We have developed a rolling circle amplification-procedure as a method to enhance signals of immunoassay (immuno-RCA) to detect one of the major food allergens, ovalbumin (OVA). Immuno-RCA in combination with a fluorescent dye and a circular DNA probe prepared by intramolecular ligation easily allowed the real-time detection of OVA, and could apparently detect signals from OVA with concentrations ranging from 10(-12) to 10(-7) g/mL. Therefore, sensitive and easily handled aspects of this method would contribute to an effective signal enhancement in immunoassay for food allergen detections.

Stand-Alone Rolling Circle Amplification Combined with Capillary Electrophoresis for Specific Detection of Small RNA

Noncoding small RNAs play diverse, important biological roles through gene expression regulation. However, their low expression levels make it difficult to identify new small RNA species and study their functions, calling for the development of detection schemes with higher simplicity, sensitivity, and specificity. Herein, we reported a straightforward assay that combined the stand-alone rolling circle amplification (RCA) with capillary electrophoresis (CE) for specific and sensitive detection of small RNAs in biological samples. In order to enhance the overall reaction efficiency and simplify the procedure, RCA was not preceded with ligation, and a preformed circular probe was employed as the template for the target small RNA-primed isothermal amplification. The long RCA product was digested and analyzed by CE. Two DNA polymerases, the Phi29 and Bst, were compared for their detection performance. Bst is superior in the aspects of specificity, procedure simplicity, and reproducibility, while Phi29 leads to a 5-fold lower detection limit and is able to detect as low as 35 amol of the target small RNA. Coamplification of an internal standard with the target and employment of the RNase A digestion step allow accurate and reproducible quantification of low amounts of small RNA targets spiked into hundreds of nanograms of the plant total RNA extract with a recovery below 110% using either enzyme. Our assay can be adapted to a capillary array system for high-throughput screening of small RNA expression in biological samples. Also, the one-step isothermal process has the potential to conveniently amplify a very limited amount of the RNA samples, e.g., RNA extracted from only a few cells, inside the capillary column or on a microchip.

Numerical Simulation of the Effects of a Thermally Significant Blood Vessel on Freezing by a Circular Surface Cryosurgical Probe Compared With Experimental Data

The dynamic thermal interaction between a surface cryosurgical probe (heat sink) and an embedded cylindrical tube (heat source), simulating a thermally-significant blood vessel, has been studied. The cryoprobe was operated by liquid nitrogen while the embedded tube was perfused by water at a constant inlet temperature. Previous experimental data were obtained in a phase-changing medium (PCM) made of 30%/70% by volume mashed potato flakes/distilled-water solution. A parametric study was conducted without the embedded tube, and with flow rates of 30 ml/min and 100 ml/min in the tube, while cooling rates at the tip of the cryoprobe were maintained at −4°C/min, −8°C/min, or −12°C/min. Numerical thermal analysis was performed by ANSYS7.0 and showed good conformity to the experimental data. The results quantify the effects of these parameters on both the shape and extent of freezing obtained in the PCM. For 20 min of operation of the cryoprobe, water temperatures inside the tube remained well above the freezing point for all assumed operating conditions. Frozen volumes of the 0°C isotherm, approximating the “frozen front,” and the −40°C isotherm, representing the “lethal temperature,” were smallest for the combination of highest cooling rate at the cryoprobe and the highest flow rate in the tube, (−12°C/min and 100 ml/min). The results indicate that both the flow rates in the embedded tube, and the cooling rates applied at the cryoprobe, have similar qualitative effects on the size of the PCM frozen volumes; increasing either one will cause these volumes to decrease. Under the conditions of this study the effects of flow rate in the tube are more pronounced, however, effecting relative frozen volumes decreases by about 10–20% while those of the cooling rate at the cryoprobe are in the range of 7–14%.

Fast simulation of ultrasound images from a CT volume

The goal of our work is to propose a fast ultrasound image simulation from CT volumes. This method is based on a model elaborated by Bamber and Dickinson that predict the appearance and properties of a B-Scan ultrasound image from the distribution of point scatterers. We propose to extend this model for the standard medical ultrasound image simulation by taking into account the acoustical tissue properties (scatterer distribution) and the geometry and the specifications of the ultrasound probe (circular probe, number and size of transducers, US pulse frequency and bandwidth, etc.). Simulations have been obtained in a fairly fast computation speed and qualitatively they present most of the real ultrasound image characteristics.

Probing technique using circular motion of a microsphere controlled by optical pressure for a nanocoordinate measuring machine

A new surface probing technique using the circular motion of an optically-trapped microsphere is proposed for a nanocoordinate measuring system. The probe sphere is oscillated circularly in the plane perpendicular to the probe axis and the circular orbit of the probe sphere is monitored for the detection of the position and normal vector direction of the surface. The principle of detection is based on changes in the circular orbit of the microsphere. When the probe approaches a work surface, the orbit of the probe sphere becomes elliptical. The minor-axis length and the minor-axis angle of the ellipse are then used as parameters to detect the position and normal vector direction of the surface, respectively. In this study, the circular motion probe is shown to have a resolution of position detection of 39 nm, and the accuracy of measuring a normal vector to the surface is on the order of 3 degrees.

Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification

A simple isothermal nucleic-acid amplification reaction, primer generation–rolling circle amplification (PG–RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60°C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, ‘primers’ are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG–RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of ‘primers’ are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (∼60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays.

Development of a novel scattered triangulation laser probe with six linear charge-coupled devices (CCDs)

Abstract The objective of this paper is to develop an inexpensive, robust, and precise scattered-type triangulation laser probe system with six linear charge-coupled devices (CCDs), that can be used to measure simultaneously the distance and inclination angle of a workpiece surface. The developed system has been configured and assembled based on Scheimpflug's principle. Through the specific arrangement of the six linear CCDs, four different measurement modules for the developed system were possible, namely the standard triangulation probe module, the double triangulation probe module, the pentagon-like triangulation probe module, and an approximately circular triangulation probe module. Angular measurements of an inclined surface were possible using the double triangulation probe module. The measuring range of the developed laser probe was ±2 mm and the resolution was 2 μm/pixel. The performance tests among the different measurement modules of the developed probe and the commercial circular triangulation laser probe OTM3-03 have been carried out. The measurement uncertainty of the developed laser probe system, with respect to different surface roughness and slope, was generally smaller than ±20 μm within the measuring range using the pentagon-like triangulation probe module with the statistical methodology of skewness (Sk) and kurtosis (Ku) analysis by setting the appropriate threshold values for Sk and Ku, respectively. The developed probe has been integrated with a PC-based 3-axis micro-positioning stage to construct an automatic non-contact 3D measurement system and to digitize the 3D profile of a small complex object.

Electrochemical immunosensor of platelet-derived growth factor with aptamer-primed polymerase amplification

A new method for the determination of platelet-derived growth factor BB (PDGF-BB) was developed using an electrochemical immunosensor with an aptamer-primed, long-strand circular detection probe. Rabbit anti-human PDGF-B polyclonal antibody was immobilized on the electrode to serve as the capture antibody. The detection probe was synthesized via polymerase extension along a single-stranded circular plasmid DNA template with a primer headed by the anti-PDGF-B aptamer. In the presence of the analyte, the aptamer-primed circular probe was captured on the electrode via the formation of an antibody/PDGF-BB/aptamer sandwiched complex. The electroactivity indicator methylene blue was adsorbed on the electrode surface via the analyte-sandwiched complex with long-strand circular DNA, thus yielding a strong electrochemical signal for the quantification of PDGF-BB. This strategy allowed electrochemical detection with enormous signal amplification arising from the long-strand localized circular probe. The oxidation peak current of methylene blue in square wave voltammetric measurements showed a linear dependence on the concentration of PDGF-BB in the range from 50 to 500 ng mL −1, with a detection limit as low as18 pg mL −1.

Visualization of DNA complexes with HMGB1 and its C-truncated form HMGB1(A+B)

Circular dichroism and scanning probe microscopy were used to characterize the interaction of DNA with the nonhistone chromatin protein HMGB1 and its recombinant version HMGB1(A+B) devoid of the C-terminal acidic region. The AFM data corroborate the earlier suggestion concerning the action of tandem DNA-binding domains, and support a modulatory role of the C-terminal domain in HMG protein-DNA interactions.

Performance of a 200‐MHz cryogenic RF probe designed for MRI and MRS of the murine brain

A cryogenic 200-MHz RF transmit/receive probe (CryoProbe) made of copper and designed for murine brain studies is described. The probe operates at 30K and its performance was compared to a home-made surface coil of equal dimensions and a mouse head volume resonator, both operating at room temperature (RT). Since for small volumes of tissue interrogated (<5 mL) sample noise and thermal noise of the receiver are of comparable magnitude, considerable noise reduction can be achieved by cooling of the receiver system, e.g., using cold helium gas. The effect of temperature lowering on the signal-to-noise ratio (SNR) was assessed in phantom and in vivo murine brain studies using conventional MRI sequences (spin and gradient echo) and single voxel MR spectroscopy. Consistent sensitivity increases were observed for the CryoProbe. SNR increases by a factor>or=2 for the phantom experiments and by factors of 1.8 to 1.9 for in vivo experiments when compared to the RT surface coil for both MRI and MRS experiments. These results are in good agreement with estimated SNR gains for a circular surface coil probe cooled to 30K. The use of a cryogenic probe represents an economically attractive option to enhance the sensitivity in small animal MRI/MRS.

3-D Eddy-Current Imaging of Metal Tubes by Gradient-Based, Controlled Evolution of Level Sets

Eddy-current nondestructive testing is widely used to detect defects within a metal structure. It is also useful to characterize their location and shape provided that proper maps of variations of impedance that the defects induce are available. Imaging of void defects in the wall of a hollow, nonmagnetic metal tube, is performed herein by controlled evolution of level sets. Such data are variations of impedance data collected by a circular probe array close to the inner surface of the tube when a coil source operated at one single frequency is set along its axis at some distance from the array, both receiver and coil source being moved simultaneously. The defect zone is represented in implicit fashion as a zero level set, which is amenable to topological changes via a nonlinear iterative method that minimizes a least-square cost functional made of the difference between the measured (computer simulated) and model data. The procedure requires the rigorous calculation of the gradient of the variations of impedance, in the case of a multistatic configuration (different driver and receiver coils), a vector domain integral field formulation being used for that purpose. Numerical examples developed by a dedicated extension of the general-purpose CIVA platform show pros and cons of the approach for inner, outer, and through-wall void defects. Further comparisons present results provided by an independently developed binary-specialized method.

Electrochemical method for precise determination of wall shear rate

The convective diffusion problem near small electrodes is solved numerically using finite volume method (direct problem). The results obtained are first used to define the limits of the quasi-steady state Leveque solution for a rectangular and circular probe. After, the algorithm is integrated to an inverse sequential method in order to obtain the wall shear rate from the measurements of the limiting diffusion currents. The results are compared with the classical techniques of calculation of wall shear rate as the quasi-steady solution, transfer function methods and the Leveque solution with transient correction.

Rolling Circle Amplification and Circle-to-circle Amplification of a Specific Gene Integrated with Electrophoretic Analysis on a Single Chip

We have developed an integrated platform for rolling circle amplification (RCA) and circle-to-circle amplification (C2CA) of circular probe (padlock probe) and subsequent microchip electrophoretic detection of a specific gene on a poly(methyl methacrylate) microchip. RCA and C2CA were successfully carried out at a steady temperature of 37 degrees C in the sample well of the microchip, and their respective product was detected on the same channel of the microchip, which was prefilled with a polymer separation matrix and fluorescent dye. Using a species-specific padlock probe for bacterial pathogen V. cholerae, a 25-ng bacterial genomic DNA could be detected in less than 65 min (including RCA and microchip electrophoresis) by this platform. Stable dsDNA C2CA product of genomic DNA for V. cholerae can be detected with the introduced integrated platform. Furthermore, the usefulness of this technique for the monitoring of RCA was demonstrated. This integrated platform provides a sensitive, fast, high-throughput, and reproducible method for signal amplification and detection of the padlock probes in the same microchip and is a promising tool for highly specific gene detection strategies.

Axial capacity estimation of straight drilled shafts in expansive and high plastic clay at different seasonal installation periods

Accepted methods of predicting drilled shafts capacities require elimination of the top five feet as a non-contributing layer in the total axial capacity estimation. The research presented in this paper focused on axial capacity calculations of shafts installed in expansive soils at different seasonal periods. The void zones, i.e. non-contact zones of drilled shafts and surrounding soil are determined by using an intrusive circular rod like probe. The length of this zone and its contribution to the total capacities are then evaluated. This research suggests that the elimination of top four feet of shaft's side friction is warranted from capacity calculations of drilled shaft if the soil tests are measured in high plastic clays in ’dry’ season. Axial capacity analysis in the wet season shows that upper layers and their side friction should be included. This paper has accounted for low strength values of the clay at wet seasonal conditions and hence incorporating the upper layer in the axial capacity estimation should not be regarded as a non-conservative step. Recommendations with respect to zone lengths and shear strength measurements of the soil layers for axial capacity of drilled shafts are provided.

Vibrotactile thresholds at the fingertip, volar forearm, large toe, and heel

Thresholds for the perception of vibration vary with location on the body due to the organization of tactile channels in hairy and non-hairy skin, and variations in receptor density. This study determined vibration thresholds at four locations on the body with two different contactors so as to assist the identification of the tactile channel determining the threshold at each location. Vibrotactile thresholds at six frequencies from 8 to 250 Hz were measured on the distal phalanx of the index finger, the volar forearm, the large toe, and the heel with two contactors: (i) a 1-mm diameter circular probe with a 1-mm gap to a fixed circular surround (i.e., 7.1-mm2 excitation area), and (ii) a 6-mm diameter circular probe with a 2-mm gap to a fixed circular surround (i.e., 79-mm2 excitation area). At all frequencies and with both contactors, thresholds on the fingertip were lower than thresholds on the volar forearm, the large toe, and the heel, consistent with a greater density of mechanoreceptors at the fingertip. Thresholds with the larger contactor were lower than thresholds with the smaller contactor on the fingertip at high frequencies (63, 125, and 250 Hz), on the large toe (except at 250 Hz), on the heel (at all frequencies), and on the volar forearm at 250 Hz. It is concluded that at least two tactile channels (Pacinian from 63 to 250 Hz, and non-Pacinian from 8 to 31.5 Hz) determined vibrotactile thresholds at the fingertip, whereas non-Pacinian channels had a dominant influence on vibrotactile thresholds at the volar forearm. The role of Pacinian and non-Pacinian channels could not be confirmed at the large toe or the heel despite some evidence of spatial summation.

Anionic lipids activate group IVA cytosolic phospholipase A2 via distinct and separate mechanisms

Previously, ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2 (cPLA2alpha). In this study, we hypothesized that these anionic lipids functionally activated the enzyme by distinctly different mechanisms. Indeed, surface plasmon resonance and surface dilution kinetics demonstrated that C1P was a more potent effector than PI(4,5)P2 in decreasing the dissociation constant of the cPLA2alpha-phosphatidylcholine (PC) interaction and increasing the residence time of the enzyme on the vesicles/micelles. PI(4,5)P2, in contrast to C1P, decreased the Michaelis-Menten constant, increasing the catalytic efficiency of the enzyme. Furthermore, PI(4,5)P2 activated cPLA2alpha with a stoichiometry of 1:1 versus C1P at 2.4:1. Lastly, PI(4,5)P2, but not C1P, increased the penetration ability of cPLA2alpha into PC-rich membranes. Therefore, this study demonstrates two distinct mechanisms for the activation of cPLA2alpha by anionic lipids. First, C1P activates cPLA2alpha by increasing the residence time of the enzyme on membranes. Second, PI(4,5)P2 activates the enzyme by increasing catalytic efficiency through increased membrane penetration.

In situdetection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS

BackgroundIn situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling. Whilst short probes have several advantages over long probes (e.g. higher resolution and specificity) they carry fewer labels per molecule and therefore require higher amplification for detection. Furthermore, short probes relying only on hybridization for specificity can result in non-specific signals appearing anywhere the probe attaches to the target specimen. One way to obtain high amplification whilst minimizing the risk of false positivity is to use small circular probes (e.g. Padlock Probes) in combination with target primed rolling circle DNA synthesis. This has previously been used for DNA detection in situ, but not until now for RNA targets.ResultsWe present here a proof of principle investigation of a novel rolling circle technology for the detection of non-polyadenylated RNA molecules in situ, including a new probe format (the Turtle Probe) and optimized procedures for its use on formalin fixed paraffin embedded tissue sections and in solid support format applications.ConclusionThe method presented combines the high discriminatory power of short oligonucleotide probes with the impressive amplification power and selectivity of the rolling circle reaction, providing excellent signal to noise ratios in combination with exact target localization due to the target primed reaction. Furthermore, the procedure is easily multiplexed, allowing visualization of several different RNAs.