Proximity Probes Research Articles

Combination of DNA walker and Pb2+-specific DNAzyme-based signal amplification with a signal-off electrochemical DNA sensor for Staphylococcus aureus detection

the accurate, reliable and specific analysis of foodborne pathogenic bacteria is vital for human health and safety. Staphylococcus aureus (S. aureus), as a common bacterium, is regularly found in food, water, and other biological samples. Herein, a signal-off electrochemical DNA sensor (E-DNA sensor) was designed for the sensitive detection ofS. aureusamplified withthecombination of a dna walker and pb2+-specific dnazyme. In this work, vancomycin functionalized gold nanoclusters (Van@Au NCs) and an aptamer strand as identification units were modified at the termini of two proximity probes. upon the addition of targetS. aureus, a dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pb2+-dependent dnazyme, achieving the conversion of oneS. aureus to many intermediate dna (t) strands. then, the released t strands hybridized with methylene blue-tagged hairpin dna (h-mb) on the electrode. consequently, the conformational alteration of t strands reduced the electron transfer efficiency of mb to the electrodeinterface (signal-off). therefore, sensitive analysis of S. aureus was readily acquired within a range of 10–107 CFU/mL and a low detection limit at 1 CFU/mL. Undoubtedly, dual recognition by aptamer and vancomycin in an integrated scheme brought about a good recognition performance of S. aureus in complex samples, as well as an efficient annihilation of harmful pathogenic bacteria during the experiment.

Influence of Fluid Film Bearings with Different Axial Groove Shapes on Automotive Turbochargers: An Experimental Study

Most commercial automotive turbochargers (TC) employ semi-floating ring bearings (SFRB) with axial groove shapes. In order to bring some insights into the role played by the axial groove geometry on the dynamics of TC, this work deals with an experimental study of the rotordynamic behavior of a stock automotive turbocharger operating on SFRB with two different groove shapes, which have the same volume and width, and with the same number of grooves. The rotating machine behavior has been evaluated under different operating conditions using a test bench specially designed to analyze turbochargers. Rotordynamic (RD) characteristics of automotive turbochargers are estimated to evaluate the influence of the axial groove geometry on the machine vibratory behavior. Frequency spectra and orbital plots of the rotor are obtained from accelerometers and proximity probes mounted on the turbocharger. The comparative analysis of the vibrational behavior of automotive turbochargers running on different supporting systems allows the identification of the role played by the axial grooves on the machine rotordynamic performance. The experimental results rendered in this work permit to classify the influence of the axial groove geometry on the turbocharger rotordynamic behavior for several speed and flow conditions.

Spatially resolved cell tagging and surfaceome labeling via targeted photocatalytic decaging

<h2>Summary</h2> Spatially resolved <i>in situ</i> tagging of the cell of interest is crucial for in-depth mechanistic dissection of multicellular architectures or processes. With continuing interest in bioorthogonal photocatalytic decaging chemistry, we herein report the extracellular-targeted photocatalytic decaging system (CAT-Ex) for spatially resolved cell tagging and surface proteome profiling under living conditions. An antibody-conjugated photocatalysis system was established and extensively validated, enabling photocatalytic decaging of biotin precursors and proximal quinone methide probes on target cells. Visible-light-controlled selective cell tagging in cell mixture as well as in primary cells from tumor xenografts were demonstrated. Spatially resolved membrane proteome profiling was further achieved by coupling quinone methide decaging chemistry with CAT-Ex, revealing a potential microdomain protein cluster surrounding the endogenous HER2 receptor. Finally, we expanded our strategy to photocatalytic prodrug decaging for selective tumor cell killing, establishing CAT-Ex as a general platform for diverse photo-controlled molecular manipulations on targeted cells with spatial-temporal precision.

Double-probe atomic force microscopy for observing spatiotemporal dynamics in a photochromic thin film

Photochromic diarylethenes undergo reversible isomerization upon alternate irradiation with ultraviolet and visible light, having excellent thermal stability and durability. Thus, in combination with localized light, they are expected to exhibit intelligent functions based on light–matter composite systems at the nanometer-scale. In previous work, the formation of a clear open-ring/closed-ring boundary was observed on the surface of photochromic diarylethene thin-film crystals by simultaneously irradiating visible and ultraviolet light. To examine dynamic processes of the boundary formation, we developed a multi-probe atomic force microscope allowing simultaneous and continuous observations at different spatial positions. In this study, we developed atomic force microscopy containing two independent probe tips that allow simultaneous observation at spatially different positions. We used a unique method to control two probes in close proximity to achieve measurements at arbitrary positions. We have correlated the structural changes on the surface of photochromic thin films with the movement of the phase boundary at the crystal surface caused by photoisomerization under simultaneous broad visible and ultraviolet light irradiation. We also measured inhomogeneous expansion of nanometer-scale photoisomerizations due to local photoexcitation. This study paves the way for the elucidation of phenomena in the dynamic optical processes of crystals and for understanding versatile spatiotemporal dynamics in two- and three-dimensional optoelectronic structures within a nanometer scale.

Experimental Investigation of Cage Dynamics and Ball-Cage Contact Forces in an Angular Contact Ball Bearing

The objectives of this investigation were to experimentally examine the cage motion and ball-cage contact forces for an angular contact ball bearing (ACBB) operating under various load and speed combinations. In order to achieve the objectives, a Counter Rotating Angular Contact Ball Bearing Test Rig (CRACTR) was designed and developed such that both the races of an ACBB can be rotated simultaneously in opposite directions, providing valuable insights into the ball-cage interaction. Five proximity probes were used concurrently to measure the in-plane (radial) and out-of-plane (axial) motion for three commercially available cage designs. Two load cells were used to measure the contact forces between the balls and cage pocket. The experimental results demonstrate that high inner race speeds and high axial loads develop the smallest and most circular cage whirl motions. Additionally, cage radius, cage pocket clearance, cage pocket area and cage inertia were the prominent factors affecting the magnitude of cage whirl. Experimental results also indicate that contact forces between the ball and cage pocket increase linearly with the increased race speeds. Ball-cage contact force was shown to be different for the three cages and was primarily governed by the cage pocket area and the cage radius. Results from this investigation can be utilized to predict the correlation between the ball-cage contact forces and the race speeds, and identify the key parameters governing the stable cage motion during bearing operation.

Proximity hybridization-mediated fluorescence resonance energy transfer for highly specific detection of tumor-derived exosomes: Combining multiple exosomal surface markers

Highly specific detection of tumor-derived exosomes is of great significance to improve the accuracy of diagnosis of cancer. Herein, based on the proximity hybridization-mediated fluorescence resonance energy transfer (FRET), a novel strategy for highly specific detection of tumor-derived exosomes was proposed by combining detection of multiple exosomal surface markers. Exosomes were enriched and separated by CD63 aptamer-functionalized nanomagnetic beads; a pair of FAM-labeled proximity probes simultaneously bind to EGFR and EpCAM on exosomes surface, which could mediate the formation of a stable DNA self-assemble complex with TAMRA-labeled signal probes through proximity hybridization, and then trigger the FRET between FAM and TAMRA to achieve highly specific detection of exosomes co-expressed CD63/EGFR/EpCAM with a LOD of 400 particles/μL. The relative standard deviation was lower than 7.3% in 50% UC-FBS due to the ratiometric signal improving its anti-interference ability. Importantly, in addition to effectively distinguish exosomes derived from A549 cells and BEAS-2B cells, the feasibility of exosomes detection in human serum was verified, and the level of exosomes co-expressed CD63/EGFR/EpCAM in non-small cell lung cancer patients (n = 15) was significantly higher than that of healthy people (n = 15) (P < 0.001). Moreover, this strategy was universal for the detection of other tumor-derived exosomes just by replacing the corresponding aptamers.

Direct Detection of Foodborne Pathogens via a Proximal DNA Probe-Based CRISPR-Cas12 Assay.

Foodborne pathogens can cause illnesses. Existing tools for detecting foodborne pathogens are typically time-consuming or require complex protocols. Here, we report an assay to directly analyze pathogenic genes based on CRISPR-Cas12. This new test, termed proximal DNA probe-based CRISPR-Cas12 (PPCas12), facilitates the detection of foodborne pathogens without amplification steps. The elimination of the nucleic acid amplification process dramatically reduced the processing time, complexity, and costs in the analysis of foodborne pathogens. The substitution of the frequently used dually labeled DNA reporter with a proximal DNA probe in the PPCas12 assay led to a 4-fold sensitivity enhancement. PPCas12 offered a limit of detection of 619 colony-forming units in the detection of Salmonella enterica (S. enterica) without the nucleic acid amplification process. The specific recognition of genes via PPCas12 allowed distinguishing S. enterica from other foodborne pathogens. The PPCas12 assay was applied in the screening of S. enterica contamination on fresh eggs with high precision. Hence, the new PPCas12 assay will be a valuable tool for on-site monitoring of foodborne pathogens.

Regenerable electrochemical biosensor for exosomes detection based on the dual-recognition proximity binding-induced DNA walker

Exosomes are secreted in the early stage of cancer at low levels that are difficult to detect due to the complex interferents of the sample matrix. Improved cancer detection requires the development of an accurate, reliable, and reusable method to assay exosomes. Here, a regenerable electrochemical biosensor was developed for exosomes detection based on the dual-recognition proximity binding-induced DNA walker and on-off-on strategy. First, we designed two proximity probes, which composed of a Pb2+-dependent DNAzyme tail sequence and identifying units (cholesterol and aptamer). By integrating two proximity probes with target exosomes, a dual-recognition proximity binding-induced DNA walker was achieved to convert one exosome to abundant intermediate DNA strands. Second, the conformational change of hairpin DNA (H) was observed with the hybridization of intermediate DNA, resulting in the increment of the distance between electroactive tag and electrode surface (“off” state of the sensor). These signal changes allowed the quantitative measurement of exosomes. Exonuclease treatment resulted in the hydrolysis of the intermediate DNA and the original hairpin structure, restoring this sensor to the “on” state. With the accurate identification of dual-recognition, the perfect signal amplification of DNA walker and the satisfactory reproducibility of an on-off-on strategy, quantitative detection of exosomes was realized in a wide range from 5.0 × 104 to 1 × 108 particles/mL, with a detection limit of 1.6 × 104 particles/mL. Overall, this approach illustrated a promising potential for exosomes distinguishment of cancer cells from normal cells, even in the complex sample matrix.

Optimising The Reference Point Within A Journal Bearing Using Laser Alignment

This paper observes different alignment conditions in a journal-bearing-supported machine (i.e. propeller bearings within the stern tube of a marine vessel). A test rig is built so that the driven machine has fixed supports, roller bearings in an electrical motor. An alignment tolerance is met at the coupling of the journal, which is positioned within three journal bearings. This research can be applied to any industry with machines of the same characteristic. The journal bearings are designed to simulate the large tolerance seen in a vessel’s stern tube only for lubrication control oil is used for the bush bearings. Various sensors are placed on the test rig to record data for later machine analysis. Using proximity probes and accelerometers the vibration of the journals and ball bearings can be visualised using orbital plotting and Fast Fourier Transform (FFT) through both LabVIEW and MATLAB respectively. Four alignment procedures are applied to the coupling using a laser unit. The first two will be so that the offset and angular misalignment is produced in the FFT spectrums as per ISO10816 Mechanical vibration so that a reference point is formed for the test rig. The other two alignment procedures represent current industry practice. These procedures are then compared to see which is the preferred reference point that will optimise the reliability value of a whole system. Finding the procedure with minimal vibration will increase the design life of housing structures for such machines, which will increase the overall profit of the shipping industry. Our developments and procedures will be further utilized in practical classes to improve maritime engineering education.

Selection and Optimization of Sensors for Monitoring of Francis Turbines

With the current demand of flexibility to hydraulic turbines, these are working under off-design conditions more than ever. Under these conditions different complex hydraulic phenomena appear, especially in Francis turbines, affecting the machine stability as well as reducing the useful life of its components. Hence, it is desirable to detect in real-time these hydraulic phenomena to assess the operation of the machine. In addition, the position and type of sensor to detect these phenomena is a key point for the monitoring of Francis units. In this paper, a large medium-head Francis turbine was instrumented with several sensors such as accelerometers, proximity probes, strain gauges, pressure sensors and a microphone. Results obtained with the different sensors in different locations are compared to know which hydraulic phenomenon is detected with every sensor. Furthermore, different signal analysis techniques are also compared. With this information, monitoring systems can be optimized with the most convenient sensors, locations and signal analysis techniques, reducing its difficulty to install and economic cost.

Transient Analysis of Pelton Turbine Prototypes

One of the most critical operating conditions in hydraulic turbines is the start-up transient. This may be characterized by the presence of several resonances, which increase the vibration and stress levels on the runners. At present, its study is of interest due to the more frequent starts and stops of the turbines in the last years. The present study is focused on analysing the start-up transient of Pelton turbines. To do so, an experimental investigation was carried out in a horizontal shaft prototype unit. The procedure started by determining the natural frequencies and mode shapes of the turbine by means of impact testing. Several accelerometers were placed on the runner, on the shaft and on the bearings. With experimental modal analysis the main vibration modes of the rotor and the runner were found. Once the structural response of the turbine was determined, the start-up transient was studied. For the tests, different types of sensors were installed on the machine. Accelerometers, proximity probes and acoustic emission sensors were placed on the bearings and on the turbine casing. Moreover, an accelerometer and a strain gauge were installed on the shaft with an on-board system. Different situations are identified during the start-up. At the very beginning of the transient, the initial collision of the water jet on the still runner produces large vibration levels due to the excitation of many bucket modes. Once the turbine starts rotating, the vibration levels increase every time the jet excitation at the bucket passing frequency meets a natural frequency of the structure. The effect of the transient regarding the change in the vibration levels and runner deformations is presented in this paper. The sensitivity of the different sensors to measure the runner vibrations is analysed in order to optimize the detection of the transient effects. The best locations to install the sensors are also identified.

A Compact Cavity-Backed Tri-Band Antenna Design for Flush Mount GNSS (L1/L5) and SDARS Operations

This letter presents a unique compact cavity-backed tri-band antenna design for supporting simultaneous Global Navigation Satellite System (GNSS) ( L 1/ L 5) and Satellite Digital Audio Radio Service (SDARS) operations in a rooftop flush mount configuration. The cavity dimensions are 100 mm in diameter and only 9.35 mm in depth to meet a specific automobile manufacturer's physical dimension requirement. The achieved half-power realized gain bandwidths at L1, L5, and SDARS bands are 85, 54, and 250 MHz, respectively. This design consists of a bottom feeding board, a top GNSS board, and a middle SDARS board. All three boards are connected using vertical wires. The left-hand circular polarization (LHCP) SDARS element is a C-line coupled circular patch element that is excited via a novel traveling-wave proximity-coupling method that maximizes the passage of the out-of-band signals. The right-hand circular polarization (RHCP) dual-ring GNSS element is excited after the SDARS element using two orthogonal tapered-width proximity probes which are connected to two microstrip lines with different lengths for producing 90° phase difference at 1.4 GHz. The entire antenna assembly is connected to a single subminiature version A (SMA) connector. The design specifications and operating principles of the proposed antenna are discussed along with comparisons between the simulation and measurement performance obtained from a fabricated prototype.

Modification of an NMR probe for monitoring of photoreactions

We describe a modified NMR probe for the in situ irradiation studies of photochemical reactions in solution-state NMR. To build up this setup, we designed an irradiation insert that brings eight light-emitting diodes (LEDs) into the NMR probe in the immediate proximity of the sample. The inserts with LEDs of different wavelengths are easily exchangeable within minutes. A tunable power supply allows to adjust the light intensity to optimize the irradiation conditions.

Experimental and Theoretical Investigation of Quasi-Static System Level Behavior of Planetary Gear Sets

Abstract This study presents a unique experimental methodology that synchronously measures various quasi-static responses of a simple four-planet planetary gear set, namely, planet load sharing, overall transmission error (OTE), and floating sun gear orbits. Strain gauges mounted directly on the planet pins were used to monitor the load shared among the planets, which is a crucial design criterion for durability and performance. High-precision optical encoders were used to measure the OTE of the gear set to explore its diagnostic value in identifying system errors. Radial motions of the floating sun gear, which are critical to the self-centering and load sharing behavior of the gear set, were monitored using magnetic proximity probes. The influence of various design parameters and operating conditions such as planet mesh phasing, carrier pin position errors, gear tooth modifications, and input torque on the system’s response will be investigated by performing an extensive set of experiments in a repeatable and accurate manner. Finally, these experimental results will be recreated theoretically using the static planetary load distribution model of Hu et al. (2018, “A Load Distribution Model for Planetary Gear Sets,” ASME J. Mech. Des., 140(5), p. 53302) to not only validate the model but also comprehend the measured behavior.

EUS-guided hepaticogastrostomy for management of cholangitis, hepatolithiasis, and anastomotic stricture after Roux-en-Y hepaticojejunostomy.

Video 1EUS-guided hepaticogastrostomy for management of cholangitis, hepatolithiasis, and anastomotic stricture after Roux-en-Y hepaticojejunostomy.

Characterization of Locus Specific Chromatin Structure and Dynamics using Correlative Conventional Super Resolution Crispr dCas9/Ms2 Imaging

Chromatin undergoes dynamic rearrangements at the nucleosomal level that influences large scale genome organization. The characterization of both chromatin structure and dynamics has been key to study mechanisms of genome organization and its influence on gene regulation. Recently, CRISPR dCas9 based photo-activation localization microscopy (PALM) has been employed to resolve nucleosomal chromatin in fixed cells. However, the fast movement of DNA and long data acquisition time associated with PALM precludes the ability to obtain locus-specific structural and dynamic information in living cells. Furthermore, the highly variable mobility distribution of DNA and bound fluorescent dCas9 probes overlaps with the one of unbound dCas9, which makes it difficult to identify dCas9 molecules bound to chromatin based on their mobility alone. Here, we develop correlative conventional and PALM imaging to identify bound dCas9 molecules while correcting for the motion of a locus to obtain structural and dynamic information that would not be accessible with traditional PALM. in addition, we employ targeted photo-activation PALM via a digital mirror device to only photoactivate dCas9 probes in proximity to a locus. by taking advantage of the difference in interaction properties between bound and unbound probes, background localizations of non-interacting probes are dramatically reduced. We demonstrate with telomeres as a model system how the combination of these techniques makes it possible to obtain both structural and dynamic information that reveals novel quantitative information about chromatin structure, compaction, and condensation that would otherwise not be obtainable using existing imaging techniques.

Experiment of the Influence of Dynamic Balancing Precision on Rotor Dynamic Behavior of 75KW Oil-free Turbo Blower

The principle and structure of the 75kW oil-free turbo blower is briefly introduced and the performance of gas foil bearings (GFBs) is tested. According to the test results, the lift-off speed of the gas foil journal bearings (GFJBs) using in 75kW oil-free turbo blower is about 4000rpm, and the gas foil thrust bearings (GFTBs) can provide at least 140N and 200N load capacity at 7200rpm and 9000rpm, respectively. The rotordynamics behavior of oil-free turbo blower is studied. Two proximity probes and speed sensor are installed to monitor the horizontal vibration displacement, vertical vibration displacement and shaft speed, respectively. And the vibration spectrum and the rotor axis orbit are analyzed. It was found that the maximum vibration amplitude of the rotor with dynamic balancing precision of G-1 was only 3.5 μm, and no other low frequency other than low frequency caused by signal interference. The fundamental frequency vibration amplitude of the rotor with dynamic balancing precision of G-2.5 is larger when running stably, but there is no low frequency and high frequency vibration with higher amplitude. Shaft couldn’t lift-off with dynamic balancing precision of G-6.3 In general, oil-free turbo blower with two kinds of dynamic balancing precision rotors can operate well.

Proximity ligation assay: an ultrasensitive method for protein quantification and its applications in pathogen detection.

It is of great significance to establish sensitive and accurate pathogen detection methods, considering the continuous emergence or re-emergence of infectious diseases seriously influences the safety of human and animals. Proximity ligation assay (PLA) is developed for the sensitive protein detection and also can be used for the detection of pathogens. PLA employs aptamer or monoclonal/polyclonal antibody-nucleic acid complexes as proximity probes. When the paired proximity probes bind to the same target protein or protein complex, they will be adjacent to each other and form an amplifiable DNA sequence through ligation. Combining the specificity of enzyme-linked immunosorbent assay (ELISA) and sensitivity of polymerase chain reaction (PCR), PLA transforms the detection of protein into the detection of DNA nucleic acid sequence. Therefore, as an ultrasensitive protein assay, PLA has great potential for quantification, localization of protein, and clinical diagnostics. In this review, we summarize the basic principles of PLA and its applications in pathogen detection. KEY POINTS: • Different forms of proximity ligation assay are introduced. • Applications of proximity ligation assay in pathogen detection are summarized. • Proximity ligation assay is an ultrasensitive method to quantify protein and pathogen.

Mathematical modeling of vibration failure caused by balancing effect in hydraulic turbines

This article discusses the causes of vibrations depending on unbalanced hydraulic forces in rotating and non-rotating equipment in hydraulic plants within the scope of vibration measurement standards. The radial and axial positions of the turbine shaft, normal operating conditions, the absolute vibration of the signal output velocity during startup and shutdown during unbalance condition of the turbine, the proximity probes have been measured by TMO 793 accelerometer sensor and TMO 793 velocity sensor. The results obtained from the monitoring are analyzed and the first discussion is over the comparison of the current operating levels and vibration standards. Secondly, the comparison is analyzed in order to examine the improvement in vibration level of the elimination of hydraulic imbalance more comprehensively, considering the stability of the hydro turbine-generator unit, a proportional row damping force, proportional-order oil-film force and a hydraulic force and by creating a new non-linear proportional-order mathematical model. Changes in vibration levels and spectral bands for failure types are stated as well. The solution of this integrated and accurate mathematical model shows that it is useful and concise in the diagnosis and prediction of faults in hydropower plants.

Simulating application of operational modal analysis to a test rig

Industrial machinery is often equipped with proximity probes that measure continuously position and vibrations of the shaft, generally relative to the bearings. These measurements are used for surveillance of the machine, and furnish valuable information to be used in a diagnostic process, especially when run-up or run-down transients are recorded and processed. However, a question arises, during normal operating conditions at constant rotating speed can some additional information about the dynamical behavior of the rotating shaft be extracted from these measurements? Considering that in an industrial environment the shaft vibrations are generally affected by some noise transmitted through the supporting structure by other machines operating in the considered industrial plant, where also fluids are moving in pipes and vessels, all generating vibrations, operational modal analysis (OMA) could be used for identifying natural frequencies and modal damping. And which are the conditions of the vibration measurements required to get reliable results from processing the recorded signals? In this context, the present paper aims to analyze, with the aid of a test rig, the conditions in which OMA will result successful. These conditions include both the operating condition of the shaft and the amount and quality of the noise. The test rig, where also noise has been applied to the rotating shaft by means of a magnetic actuator, furnishes some test runs that are analyzed and used for tuning a model that can then be used to explore the different conditions.