Proximity Probes Research Articles

Proximity ligation assay to detect and localize the interactions of ERα with PI3-K and Src in breast cancer cells and tumor samples.

In situ proximity ligation assay (PLA) is a powerful method for detection, localization, and quantification of proteins, protein-protein interactions, and posttranslational modifications. Proteins detected by two specific antibodies are recognized by proximity probes conjugated with complementary oligonucleotides to allow the formation of circular DNA probes when bound in close proximity. Subsequent amplification of this DNA can then be visualized. Here, we describe the in situ PLA method for the detection of the ERα/Src/PI3K complex in breast cancer. We used two different techniques for detecting the signals: fluorescent detection for cell line analysis and bright-field revelation, which is better suited to clinical analysis of patient samples.

Atomic Force Microscope Controlled Topographical Imaging and Proximal Probe Thermal Desorption/Ionization Mass Spectrometry Imaging

This paper reports on the development of a hybrid atmospheric pressure atomic force microscopy/mass spectrometry imaging system utilizing nanothermal analysis probes for thermal desorption surface sampling with subsequent atmospheric pressure chemical ionization and mass analysis. The basic instrumental setup and the general operation of the system were discussed, and optimized performance metrics were presented. The ability to correlate topographic images of a surface with atomic force microscopy and a mass spectral chemical image of the same surface, utilizing the same probe without moving the sample from the system, was demonstrated. Co-registered mass spectral chemical images and atomic force microscopy topographical images were obtained from inked patterns on paper as well as from a living bacterial colony on an agar gel. Spatial resolution of the topography images based on pixel size (0.2 μm × 0.8 μm) was better than the resolution of the mass spectral images (2.5 μm × 2.0 μm), which were limited by current mass spectral data acquisition rate and system detection levels.

Effects of slurry filling and mill speed on the net power draw of a tumbling ball mill

The pool of slurry is known to lower the power drawn to the mill. An attempt to ascertain this observation by relating load orientation to mill power for a range of speeds and slurry fillings was undertaken.To this end, a Platinum ore (−850μm) was used to prepare a slurry at 65% solids concentration by mass. The Wits pilot mill (552×400mm), initially loaded with 10mm balls at 20% volumetric filling, was run at 5 different speeds between 65% and 85% of critical. The net power draw and media charge position were measured. After this, the slurried ore was gradually added to the media charge for slurry filling U between 0 and 3. A proximity probe and a conductivity sensor mounted on the mill shell provided a means of measuring both the position of the media charge and that of slurry. The data collected for the load behaviour and net power draw was later analysed.It was found that Morrell’s model could not fully explain the effect of slurry volume on net power draw especially for an under-filled media charge (i.e., for U<1). The size of lifters and grinding balls used could be the reason for this. That is why a piece-wise function was curve-fitted to the power data to help make sense of the inconsistencies observed.

A simple and portable device for the quantification of TNF-α in human plasma by means of on-chip magnetic bead-based proximity ligation assay

There is a general need in healthcare systems all around the world to reduce costs in terms of time and money without compromising patients outcome. Point-of-Care Testing (POCT) is currently being used in some applications (e.g. POC coagulation devices) as an alternative to already established standard central laboratory tests to overcome sample transportation and long turnaround times. The main objective of this investigation was to quantify Tumour Necrosis Factor-alpha (TNF-α) on-chip within the clinical relevant range of 5–100pg/mL in human pooled plasma. The novel solid-phase assay developed in this study was a magnetic bead-based proximity ligation assay (PLA) in which one of the assay proximity probes was directly immobilised onto streptavidin-coated magnetic beads. The portable device was based on a disposable and single-use cyclo-olefin polymer (COP) microfluidic chip interfaced with a quantitative real-time polymerase chain reaction (qPCR) device previously developed in-house. Sample volume was 10µL and total assay time under 3h. The POC device and assay developed offer portability, smaller reagent and sample consumption, and faster time-to-results compared with standard ELISAs. Determination and monitoring of TNF-α therapy at the point-of-care will help to improve clinical and/or economical outcome in governmental healthcare budgets.

Proximity-dependent protein detection based on enzyme-assisted fluorescence signal amplification

In this paper, we develop a sensitive fluorescence method for protein detection based on proximity extension and enzyme-assisted signal amplification. In this novel method, pairs of proximity probes are designed, and the recognition elements are integrated into the proximity probes. Then proteins are detected by transforming aptamer or antibody-protein binding signals into DNA detection based on proximity effect. In addition, nick sites are introduced into the proximity probes to amplify the detectable signal. As proof of concept, detection of human α-thrombin and human IgG are demonstrated in this study. The aptamers and antibodies are coupled in the proximity probes as recognition elements for human α-thrombin and human IgG respectively. In the presence of target protein, aptamer or antibody-protein binding signals are transformed into detectable signals by the proximity effect, and can be further amplified by enzyme-assisted strand displacement. The above mentioned strategies consequently bring the limit of detection (LOD) to as low as 1pM for human α-thrombin and 6pM for human IgG. Furthermore, this method might be extended to sensitive detection of other proteins by changing recognition elements of proximity probes.

Proximity-dependent isothermal cycle amplification for small-molecule detection based on surface enhanced Raman scattering

A novel proximity-dependent isothermal cycle amplification (PDICA) strategy has been proposed and successfully used for the determination of cocaine coupled with surface enhanced Raman scattering (SERS). For enhancing the SERS signal, Raman dye molecules modified bio-barcode DNA and gold nanoparticles (AuNPs) are used to prepare the Raman probes. Magnetic beads (MBs) are used as the carrier of amplification template and signal output products for circumventing the problem of high background induced by excess bio-barcode DNA. In the presence of target molecules, two label-free proximity probes can hybridize with each other and subsequently opens the hairpin connector-probe to perform the PDICA reaction including the target recycling amplification and strand-displacement amplification. As a result, abundant AuNPs Raman probes can be anchored on the surface of MBs and a low detection limit of 0.1nM for cocaine is obtained. This assay also exhibits an excellent selectivity and has been successfully performed in human serum, which confirms the reliability and practicality of this protocol.

Pharyngeal pH monitoring better predicts a successful outcome for extraesophageal reflux symptoms after antireflux surgery

Gastroesophageal reflux disease can be associated with extraesophageal symptoms (hoarseness, cough, asthma, and globus). However, these symptoms may have a multifactorial etiology. Proximal pH monitoring has been proposed as a means of identifying patients where reflux is the cause of the extraesophageal symptoms. The aim of this study was to determine whether proximal esophageal or pharyngeal pH monitoring better identified patients with extraesophageal symptoms that improved after antireflux surgery. A retrospective chart review was performed to identify all patients who had esophageal and pharyngeal pH monitoring before an antireflux operation. A composite score was used to define an abnormal result with each test. A successful outcome was defined as improvement or resolution of extraesophageal symptoms. There were 20 patients identified. Antireflux surgery led to a successful outcome in 14 patients (70 %). Restech better identified patients with extraesophageal symptoms who had a successful outcome with antireflux surgery (12 of 14 [86 %] based on abnormal Restech versus 5 of 10 [50 %] based on abnormal proximal probe, p = 0.06). Comparing only the 15 patients who had both proximal esophageal and pharyngeal pH monitoring, Restech again better identified those who had a successful outcome with antireflux surgery (9 of 10 [90 %] based on abnormal Restech versus 5 of 10 [50 %] based on abnormal proximal probe, p = 0.05). The positive and negative predictive values for symptomatic improvement after a fundoplication were better for an abnormal Restech score than for an abnormal proximal esophageal score (80 vs. 71 % and 60 vs. 38 %, respectively). In two patients with a successful outcome, Restech was the only positive test. In patients with extraesophageal reflux symptoms, proximal esophageal pH monitoring failed to identify half of the patients who had a successful outcome after antireflux surgery. In contrast, an abnormal Restech pH test was present in 90 % of patients with a successful outcome. Further, a negative Restech study more reliably indicated the absence of reflux-induced extraesophageal symptoms. Our results indicate that Restech pharyngeal pH monitoring should be utilized in the evaluation of patients with extraesophageal symptoms that may be associated with reflux disease.

Inspired gas humidity and temperature during mechanical ventilation with the Stephanie ventilator

To measure inspired gas humidity and temperature delivered by a Stephanie neonatal ventilator with variations in (i) circuit length; (ii) circuit insulation; (iii) proximal airway temperature probe (pATP) position; (iv) inspiratory temperature (offset); and (v) incubator temperatures. Using the Stephanie neonatal ventilator, inspired gas humidity and temperature were measured during mechanical ventilation at the distal inspiratory limb and 3 cm down the endotracheal tube. Measurements were made with a long or short circuit; with or without insulation of the inspiratory limb; proximal ATP (pATP) either within or external to the incubator; at two different inspiratory temperature (offset) of 37(-0.5) and 39(-2.0)°C; and at three different incubator temperatures of 32, 34.5, and 37°C. Long circuits produced significantly higher inspired humidity than short circuits at all incubator settings, while only at 32°C was the inspired temperature higher. In the long circuits, insulation further improved the inspired humidity especially at 39(-2.0)°C, while only at incubator temperatures of 32 and 37°C did insulation significantly improve inspired temperature. Positioning the pATP outside the incubator did not result in higher inspired humidity but did significantly improve inspired temperature. An inspiratory temperature (offset) of 39(-2.0)°C delivered significantly higher inspired humidity and temperature than the 37(-0.5)°C especially when insulated. Long insulated Stephanie circuits should be used for neonatal ventilation when the infant is nursed in an incubator. The recommended inspiratory temperature (offset) of 37(-0.5)°C produced inspired humidity and temperature below international standards, and we suggest an increase to 39(-2.0)°C.

Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue

Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.

Active vibrations control of journal bearings with the use of piezoactuators

Rotor instability is one of the most serious problems of high-speed rotors supported by sliding bearings. With constantly increasing parameters, new machines problems with rotor instability are encountered more and more often. Even though there are many solutions based on passive improvement of the bearing geometry to enlarge the operational speed range of the journal bearing, the paper deals with a working prototype of a system for the active vibration control of journal bearings with the use of piezoactuators. The controllable journal bearing is a part of a test rig, which consists of a rotor driven by an inductive motor up to 23,000rpm. The actively controlled journal bearing consists of a movable bushing, which is actuated by two piezoactuators. The journal vibration is measured by a pair of proximity probes. The control system enables run-up, coast-down and steady-state rotation. A real-time simulator dSpace encloses the control loop. Force produced by piezoactuators and acting at the bushing is controlled according to error signals derived from the proximity probe output signals. As it was proved by experiments the active vibration control extends considerably the range of the operational speed.

A Compact Dual-Band GPS Antenna Design

This letter discusses a small slot-loaded, proximity-fed patch antenna designed for GPS operation at L1 (1575 MHz) and L2 (1227 MHz) bands. High-dielectric substrate and meandered slots are employed to reduce the antenna size down to 25.4 mm in diameter and 11.27 mm in thickness. The thickness is important for achieving the wide bandwidth (>30 MHz) in support of modern GPS coding schemes. The dual-band coverage is achieved by utilizing the patch mode in L2 band and slot mode in L1 band. This design features additional slot stubs for independently tuning the L1 frequency. The right-hand circularly polarized (RHCP) field property is achieved by connecting two proximity probes to a small surface-mount 0°-90° hybrid chip. Simulated and measured antenna performance will be presented. This compact GPS antenna design is suitable for small GPS antenna arrays and portable GPS devices.

Design of a LabVIEW System Applied to Predictive Maintenance

The implementation of vibration analysis techniques based on virtual instrumentation has spread increasingly in the academic and industrial branch, since the use of any software for this type of analysis brings good results at low cost. Among the existing software for programming and creation of virtual instruments, the LabVIEW was chosen for this project. This software has good interface with the method of graphical programming. In this project, it was developed a system of rotating machine condition monitoring. This monitoring system is applied in a test stand, simulating large scale applications, such as in hydroelectric, nuclear and oil exploration companies. It was initially used a test stand, where an instrumentation for data acquisition was inserted, composed of accelerometers and inductive proximity sensors. The data collection system was structured on the basis of an NI 6008 A/D converter of National Instruments. An electronic circuit command was developed through the A/D converter for a remote firing of the test stand. The equipment monitoring is performed through the data collected from the sensors. The vibration signals collected by accelerometers are processed in the time domain and frequency. Also, proximity probes were used for the axis orbit evaluation and an inductive sensor for the rotation and trigger measurement.

Domain Wall Geometry Controls Conduction in Ferroelectrics

A new paradigm of domain wall nanoelectronics has emerged recently, in which the domain wall in a ferroic is itself an active device element. The ability to spatially modulate the ferroic order parameter within a single domain wall allows the physical properties to be tailored at will and hence opens vastly unexplored device possibilities. Here, we demonstrate via ambient and ultrahigh-vacuum (UHV) scanning probe microscopy (SPM) measurements in bismuth ferrite that the conductivity of the domain walls can be modulated by up to 500% in the spatial dimension as a function of domain wall curvature. Landau-Ginzburg-Devonshire calculations reveal the conduction is a result of carriers or vacancies migrating to neutralize the charge at the formed interface. Phase-field modeling indicates that anisotropic potential distributions can occur even for initially uncharged walls, from polarization dynamics mediated by elastic effects. These results are the first proof of concept for modulation of charge as a function of domain wall geometry by a proximal probe, thereby expanding potential applications for oxide ferroics in future nanoscale electronics.

Reciprocity Theory of Apertureless Scanning Near-Field Optical Microscopy with Point-Dipole Probes

Near-field microscopy offers the opportunity to reveal optical contrast at deep subwavelength scales. In scanning near-field optical microscopy (SNOM), the diffraction limit is overcome by a nanoscopic probe in close proximity to the sample. The interaction of the probe with the sample fields necessarily perturbs the bare sample response, and a critical issue is the interpretation of recorded signals. For a few specific SNOM configurations, individual descriptions have been modeled, but a general and intuitive framework is still lacking. Here, we give an exact formulation of the measurable signals in SNOM which is easily applicable to experimental configurations. Our results are in close analogy with the description Tersoff and Hamann have derived for the tunneling currents in scanning tunneling microscopy. For point-like scattering probe tips, such as used in apertureless SNOM, the theory simplifies dramatically to a single scalar relation. We find that the measured signal is directly proportional to the field of the coupled tip-sample system at the position of the tip. For weakly interacting probes, the model thus verifies the empirical findings that the recorded signal is proportional to the unperturbed field of the bare sample. In the more general case, it provides guidance to an intuitive and faithful interpretation of recorded images, facilitating the characterization of tip-related distortions and the evaluation of novel SNOM configurations, both for aperture-based and apertureless SNOM.

Rotordynamic Performance of Shimmed Gas Foil Bearings for Oil-Free Turbochargers

Oil-free turbochargers (TCs) will increase the power and efficiency of internal combustion engines, both sparking ignition and compression ignition, without engine oil lubricant feeding or scheduled maintenance. Using gas foil bearings (GFBs) in passenger vehicle TCs enables compact, lightweight, oil-free systems, along with accurate shaft motion. This paper presents extensive test measurements on GFBs for oil-free TCs, including static load-deflection measurements of test GFBs, rotordynamic performance measurements of a compressed air driven oil-free TC unit supported on test GFBs, and bench test measurements of the oil-free TC driven by a passenger vehicle diesel engine. Two configurations of GFBs, one original and the other modified with three shims, are subjected to a series of experimental tests. For the shimmed GFB, three metal shims are inserted under the bump-strip layers, in contact with the bearing housing. The installation of shims creates mechanical preloads that enhance a hydrodynamic wedge in the assembly radial clearance to generate more film pressure. Simple static load-deflection tests estimate the assembly radial clearance of the shimmed GFB, which is smaller than that of the original GFB. Model predictions agree well with test data. The discrepancy between the model predictions and test data is attributed to fabrication inaccuracy in the top foil and bump strip layers. Test GFBs are installed into a TC test rig driven by compressed air for rotordynamic performance measurements. The test TC rotor, 335 g in weight and 117 mm long, is coated with a commercially available, wear-resistant solid lubricant, Amorphous M, to prevent severe wear during start-up and shutdown in the absence of an air film. A pair of optical proximity probes positioned orthogonally at the compressor end record lateral rotor motions. Rotordynamic test results show that the shimmed GFB significantly diminishes the large amplitude of subsynchronous rotor motions arising in the unmodified GFB. Predicted synchronous rotor amplitudes and rigid body mode natural frequencies agree reasonably well with recorded test data. Finally, the oil-free TC is installed into a passenger vehicle diesel engine test bench. The TC rotor speed is controlled by the vehicle engine. Speed-up tests show dominant synchronous motion (1X) of the rotor. Whirl frequencies of the relatively small subsynchronous motions are associated with the rigid body natural mode of the TC rotor-GFB system as well as (forced) excitation from the four-cylinder diesel engine. The bench test measurements demonstrate a significant reduction in the amplitude of subsynchronous motions for the shimmed GFB, thus verifying the preliminary test results in the TC test rig driven by compressed air.

The effect of external dynamic loads on the lifetime of rolling element bearings: accurate measurement of the bearing behaviour

Accurate prediction of the lifetime of rolling element bearings is a crucial step towards a reliable design of many rotating machines. Recent research emphasizes an important influence of external dynamic loads on the lifetime of bearings. However, most lifetime calculations of bearings are based on the classical ISO 281 standard, neglecting this influence. For bearings subjected to highly varying loads, this leads to inaccurate estimations of the lifetime, and therefore excessive safety factors during the design and unexpected failures during operation. This paper presents a novel test rig, developed to analyse the behaviour of rolling element bearings subjected to highly varying loads. Since bearings are very precise machine components, their motion can only be measured in an accurately controlled environment. Otherwise, noise from other components and external influences such as temperature variations will dominate the measurements. The test rig is optimised to perform accurate measurements of the bearing behaviour. Also, the test bearing is fitted in a modular structure, which guarantees precise mounting and allows testing different types and sizes of bearings. Finally, a fully controlled multi-axial static and dynamic load is imposed on the bearing, while its behaviour is monitored with capacitive proximity probes.

Surface thermocouples for measurement of pulsed heat flux in the divertor of the Alcator C-Mod tokamak

A novel set of thermocouple sensors has been developed to measure heat fluxes arriving at divertor surfaces in the Alcator C-Mod tokamak, a magnetic confinement fusion experiment. These sensors operate in direct contact with the divertor plasma, which deposits heat fluxes in excess of ~10 MW/m(2) over an ~1 s pulse. Thermoelectric EMF signals are produced across a non-standard bimetallic junction: a 50 μm thick 74% tungsten-26% rhenium ribbon embedded in a 6.35 mm diameter molybdenum cylinder. The unique coaxial geometry of the sensor combined with its single-point electrical ground contact minimizes interference from the plasma/magnetic environment. Incident heat fluxes are inferred from surface temperature evolution via a 1D thermal heat transport model. For an incident heat flux of 10 MW/m(2), surface temperatures rise ~1000 °C/s, corresponding to a heat flux flowing along the local magnetic field of ~200 MW/m(2). Separate calorimeter sensors are used to independently confirm the derived heat fluxes by comparing total energies deposited during a plasma pulse. Langmuir probes in close proximity to the surface thermocouples are used to test plasma-sheath heat transmission theory and to identify potential sources of discrepancies among physical models.

Externally sensitized deprotection of PPG-masked carbonyls as a spatial proximity probe in photoamplified detection of binding events

Externally sensitized electron-transfer fragmentation in dithiane PPG (photoremovable protecting group)-protected carbonyls is adopted for detection and amplification of molecular recognition events. The new methodology allows for detection of as low as 50 attomoles of avidin utilizing an imager based on a low sensitivity mass-produced consumer CCD camera. Numeric modelling is carried out to demonstrate the intrinsic limitations of 2D amplification on surfaces and the advantages of unconstrained amplification in a compartmentalized volume of spatially addressable 3D solutions.

Feasibility of Dual Wireless Esophageal pH Monitoring

Wireless pH monitoring is currently employed in the distal esophagus. There are no controlled studies on the feasibility/safety of proximal esophageal wireless pH capsule placement. We tested the hypothesis that there will be no difference in patient perception of a more proximally placed pH capsule. Randomized single-blinded sham-controlled trial. Tertiary care center. All patients had a wireless pH capsule positioned 6 cm proximal to the gastroesophageal junction. They were randomized into intervention or sham groups after distal capsule placement. The delivery introducer was positioned 10 cm proximal to the distal esophageal capsule, and a second capsule was either deployed (intervention) or not (sham) based on group allocation. Patients were blinded to group assignment. Modified Edmonton Score was used to assess for chest pain, dysphagia, and odynophagia. The primary endpoints were (1) required endoscopic removal because of discomfort and (2) change in chest pain scores, controlling for baseline pain. Patients were randomized to either the intervention, proximal esophageal capsule (n = 11), or sham (n = 11). Patients with proximal pH probes had higher odds of having their chest pain (odds ratio [OR], 8.44; 95% confidence interval [CI], 1.35-52.6; P = .02), odynophagia (OR, 49.5; 95% CI, 4.70-520; P = .001), and dysphagia (OR, 14.3; 95% CI, 2.12-96.6; P = .006) exacerbated. Two (2/11; 18%) proximally deployed probes required endoscopic removal because of patient intolerance or discomfort. A proximal esophageal wireless pH monitor placement is feasible but results in increased chest pain, odynophagia, and dysphagia that can be severe enough to require endoscopic removal. These limitations preclude its potential clinical benefit.

Sensitive Detection of Small Molecules by Competitive Immunomagnetic-Proximity Ligation Assay

A novel detection method of small molecules, competitive immunomagnetic-proximity ligation assay (CIPLA), was developed and described in this report. Through the proximity effect caused by special proximity probes we prepared, small molecules can be detected using only one monoclonal antibody. CIPLA overcomes the obstacle that the proximity ligation assay (PLA) cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. Two small molecular compounds, clenbuterol (CLE) and ractopamine (RAC), were selected as targets for CIPLA. The limit of detection (LOD) reached 0.01 ng mL(-1), which was 10-50-fold lower than ELISA. With 5 orders of magnitude of the dynamic range achieved, the excellent sensitivity and broad dynamic range of CIPLA are noted. It can be applied widely in the sensitive detection of many other small molecular materials such as pesticides, additives in food, drugs, and biological samples, which have great significance in both theoretical and practical aspects.