Robust Probe Research Articles

Phase-stepped fringe projection by rotation about the camera’s perspective center

A technique to produce phase steps in a fringe projection system for shape measurement is presented. Phase steps are produced by introducing relative rotation between the object and the fringe projection probe (comprising a projector and camera) about the camera's perspective center. Relative motion of the object in the camera image can be compensated, because it is independent of the distance of the object from the camera, whilst the phase of the projected fringes is stepped due to the motion of the projector with respect to the object. The technique was validated with a static fringe projection system by moving an object on a coordinate measuring machine (CMM). The alternative approach, of rotating a lightweight and robust CMM-mounted fringe projection probe, is discussed. An experimental accuracy of approximately 1.5% of the projected fringe pitch was achieved, limited by the standard phase-stepping algorithms used rather than by the accuracy of the phase steps produced by the new technique.

CdSe/CdS Semiconductor Quantum Rods as Robust Fluorescent Probes for Paraffin-Embedded Tissue Imaging

Immunofluorescence techniques on formalin fixed paraffin-embedded sections allow for the evaluation of the expression and spatial distribution of specific markers in patient tissue specimens or for monitoring the fate of labeled cells after in vivo injection. This technique suffers however from the auto-fluorescence background signal of the embedded tissue that eventually confounds the analysis. Here we show that rod-like semiconductor nanocrystals (QRs), intramuscularly injected in living mice, could be clearly detected by confocal microscopy in formalin fixed paraffin-embedded tissue sections. Despite the low amount of QRs amount injected (25 picomoles), these were clearly visible after 24 h in the muscle sections and their fluorescence signal was stronger than that of CdSe/ZnS quantum dots (QDs) similarly functionalized and in the case of QRs only, the signal lasted even after 21 days after the injection.

Label-free optical bifunctional oligonucleotide probe for homogeneous amplification detection of disease markers

Oligonucleotide-based detection schemes that avoid chemical modification possess significant advantages, including simplified design, intrinsic affinity for targets, low cost and ease to extend applications. In this contribution, we developed a label-free self-locked bifunctional oligonucleotide probe (signaling probe) for the detection of different disease markers in parallel. Two signal enhancement techniques based on isothermal circular strand-displacement polymerization reaction, cyclical nucleic acid strand-displacement polymerization (CNDP) and cyclical common (nonnucleic acid) target-displacement polymerization (CCDP), were employed to implement the amplification assay for p53 gene and PDGF-BB, respectively. The attractive assay properties confirmed the effectiveness of isothermal polymerization in common biosensing systems without evolving any chemical modification: PDGF could be detected down to 0.87ng/mL, and a dynamic response range of 8–5000ng/mL was achieved; The capability to screen the p53 gene was also considerably improved, including the detection limit, sensitivity, dynamic range and so on. Moreover, because no any chemical modification of the signaling probe was acquired and different targets were separately detected in homogeneous solution. This interrogating platform exhibits the design flexibility, convenience, simplicity and cost-effectiveness. The success achieved here is expected to serve as a significant step toward the development of robust label-free oligonucleotide probes in biomarker profiling and disease diagnostics.

Rapid, Photoactivatable Turn-On Fluorescent Probes Based on an Intramolecular Photoclick Reaction

Photoactivatable fluorescent probes are invaluable tools for the study of biological processes with high resolution in space and time. Numerous strategies have been developed in generating photoactivatable fluorescent probes, most of which rely on the photo-"uncaging" and photoisomerization reactions. To broaden photoactivation modalities, here we report a new strategy in which the fluorophore is generated in situ through an intramolecular tetrazole-alkene cycloaddition reaction ("photoclick chemistry"). By conjugating a specific microtubule-binding taxoid core to the tetrazole/alkene prefluorophores, robust photoactivatable fluorescent probes were obtained with fast photoactivation (∼1 min) and high fluorescence turn-on ratio (up to 112-fold) in acetonitrile/PBS (1:1). Highly efficient photoactivation of the taxoid-tetrazoles inside the mammalian cells was also observed under a confocal fluorescence microscope when the treated cells were exposed to either a metal halide lamp light passing through a 300/395 filter or a 405 nm laser beam. Furthermore, a spatially controlled fluorescent labeling of microtubules in live CHO cells was demonstrated with a long-wavelength photoactivatable taxoid-tetrazole probe. Because of its modular design and tunability of the photoactivation efficiency and photophysical properties, this intramolecular photoclick reaction based approach should provide a versatile platform for designing photoactivatable fluorescent probes for various biological processes.

Monodisperse Magnetic Nanoparticles for Theranostic Applications

Effective medical care requires the concurrent monitoring of medical treatment. The combination of imaging and therapeutics allows a large degree of control over the treatment efficacy and is now commonly referred to as "theranostics". Magnetic nanoparticles (NPs) provide a unique nanoplatform for theranostic applications because of their biocompatibility, their responses to the external magnetic field, and their sizes which are comparable to that of functional biomolecules. Recent studies of magnetic NPs for both imaging and therapeutic applications have led to greater control over size, surface functionalization, magnetic properties, and specific binding capabilities of the NPs. The combination of the deep tissue penetration of the magnetic field and the ability of magnetic NPs to enhance magnetic resonance imaging sensitivity and magnetic heating efficiency makes magnetic NPs promising candidates for successful future theranostics. In this Account, we review recent advances in the synthesis of magnetic NPs for biomedical applications such as magnetic resonance imaging (MRI) and magnetic fluid hyperthermia (MFH). Our focus is on iron oxide (Fe(3)O(4)) NPs, gold-iron oxide (Au-Fe(3)O(4)) NPs, metallic iron (Fe) NPs, and Fe-based alloy NPs, such as iron-cobalt (FeCo) and iron-platinum (FePt) NPs. Because of the ease of fabrication and their approved clinical usage, Fe(3)O(4) NPs with controlled sizes and surface chemistry have been studied extensively for MRI and MFH applications. Porous hollow Fe(3)O(4) NPs are expected to have similar magnetic, chemical, and biological properties as the solid Fe(3)O(4) NPs, and their structures offer the additional opportunity to store and release drugs at a target. The Au-Fe(3)O(4) NPs combine both magnetically active Fe(3)O(4) and optically active Au within one nanostructure and are a promising NP platform for multimodal imaging and therapeutics. Metallic Fe and FeCo NPs offer the opportunity for probes with even higher magnetizations. However, metallic NPs are normally very reactive and are subject to fast oxidation in biological solutions. Once they are coated with a layer of polycrystalline Fe(3)O(4) or a graphitic shell, these metallic NPs are more stable and provide better contrast for MRI and more effective heating for MFH. FePt NPs are chemically more stable than Fe and FeCo NPs and have shown great potential as contrast agents for both MRI and X-ray computed tomography (CT) and as robust probes for controlled heating in MFH.

A Simple Tactile Probe for Surface Identification by Mobile Robots

This paper describes a tactile probe designed for surface identification in a context of all-terrain low-velocity mobile robotics. The proposed tactile probe is made of a small metallic rod with a single-axis accelerometer attached near its tip. Surface identification is based on analyzing acceleration patterns induced at the tip of this mechanically robust tactile probe, while it is passively dragged along a surface. A training dataset was collected over ten different indoor and outdoor surfaces. Classification results for an artificial neural network were positive, with an 89.9% and 94.6% success rate for 1- and 4-s time windows of data, respectively. We also demonstrated that the same tactile probe can be used for unsupervised learning of terrains. For 1-s time windows of data, the classification success rate was only reduced to 74.1%. Finally, a blind mobile robot, performing real-time classification of surfaces, demonstrated the feasibility of this tactile probe as a guidance mechanism.

Robustness to systematics for future dark energy probes

We extend the Figure of Merit formalism usually adopted to quantify the statistical performance of future dark energy probes to assess the robustness of a future mission to plausible systematic bias. We introduce a new robustness Figure of Merit which can be computed in the Fisher Matrix formalism given arbitrary systematic biases in the observable quantities. We argue that robustness to systematics is an important new quantity that should be taken into account when optimizing future surveys. We illustrate our formalism with toy examples, and apply it to future type Ia supernova (SNIa) and baryonic acoustic oscillation (BAO) surveys. For the simplified systematic biases that we consider, we find that SNIa are a somewhat more robust probe of dark energy parameters than the BAO. We trace this back to a geometrical alignement of systematic bias direction with statistical degeneracy directions in the dark energy parameter space.

Photoabsorption and Resonance Energy Transfer Phenomenon in CdTe−Protein Bioconjugates: An Insight into QD−Biomolecular Interactions

Luminescent quantum dots (QDs) possess unique photophysical properties, which are advantageous in the development of new generation robust fluorescent probes based on Forster resonance energy transfer (FRET) phenomena. Bioconjugation of these QDs with biomolecules create hybrid materials having unique photophysical properties along with biological activity. The present study is aimed at characterizing QD bioconjugates in terms of optical behavior. Colloidal CdTe QDs capped with 3-mercaptopropionic acid (MPA) were conjugated to different proteins by the carbodiimide protocol using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and a coupling reagent like N-hydroxysuccinimide (NHS). The photoabsorption of these QD-protein bioconjugates demonstrated an effective coupling of electronic orbitals of constituents. A linear variation in absorbance of bioconjugates at 330 nm proportionate to conjugation suggests a covalent attachment as confirmed by gel electrophoresis. A red shift in the fluorescence of bovine serum albumin (BSA) due to conjugation inferred a decrease in Stokes shift and solvent polarization effects on protein. A proportionate quenching in BSA fluorescence followed by an enhancement of QD fluorescence point toward nonradiative dipolar interactions. Further, reduction in photobleaching of BSA suggests QD-biomolecular interactions. Bioconjugation has significantly influenced the photoabsorption spectrum of QD bioconjugates suggesting the formation of a possible protein shell on the surface of QD. The experimental result suggests that these bioconjugates can be considered nanoparticle (NP) superstructures for the development of a new generation of robust nanoprobes.

Multiplex, Quantitative, Ligation-Dependent Probe Amplification for Determination of Allergens in Food

Legislation requires labeling of foods containing allergenic ingredients. Here, we present a robust 10-plex quantitative and sensitive ligation-dependent probe amplification method, the allergen-multiplex ligation-dependent probe amplification (MLPA) method, for specific detection of eight allergens: sesame, soy, hazelnut, peanut, lupine, gluten, mustard, and celery. Ligated probes were amplified by polymerase chain reaction (PCR), and amplicons were detected using capillary electrophoresis. Quantitative results were obtained by comparing signals with an internal positive control. The limit of detection varied from approximately 5 to 400 gene copies, depending on the allergen. The method was tested using different foods spiked with mustard, celery, soy, or lupine flour in the 1-0.001% range. Depending on the allergen, sensitivities were similar or better than those obtained with qPCR. The allergen-MLPA method is modular and can be adapted by adding probe pairs for other allergens. The DNA-based allergen-MLPA method will constitute a complementary method to the traditional protein-based methods.

Stable Single-Crystalline Body Centered Cubic Fe Nanoparticles

We report a facile synthesis of body centered cubic (bcc) Fe nanoparticles (NPs) via the thermal decomposition of iron pentacarbonyl, Fe(CO)(5), in the presence of hexadecylammonium chloride. These bcc-Fe NPs exhibit a drastically increased stability and magnetic moment (M(s) = 164 A·m(2)·kg(-1)(Fe)) even in physiological solutions, and have much enhanced magnetic imaging contrast (r(2) = 220 s(-1)·mM(-1)) and heating (SAR = 140 W·g(-1)(Fe)) effects. They may serve as robust probes for imaging and therapeutic applications.

Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

BackgroundWe present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity.ResultsWe developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU.ConclusionsThe described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

Microscopic Mechanism of 1/f Noise in Graphene: Role of Energy Band Dispersion

A distinctive feature of single-layer graphene is the linearly dispersive energy bands, which in the case of multilayer graphene become parabolic. A simple electrical transport-based probe to differentiate between these two band structures will be immensely valuable, particularly when quantum Hall measurements are difficult, such as in chemically synthesized graphene nanoribbons. Here we show that the flicker noise, or the 1/f noise, in electrical resistance is a sensitive and robust probe to the band structure of graphene. At low temperatures, the dependence of noise magnitude on the carrier density was found to be opposite for the linear and parabolic bands. We explain our data with a comprehensive theoretical model that clarifies several puzzling issues concerning the microscopic origin of flicker noise in graphene field-effect transistors (GraFET).

Peptide Aptamer Microarrays: Bridging the bio–detector interface

In the near future, personalised medicine and phase-0 trials will require that clinical practitioners move from the "one biomarker per disease" paradigm to the use of molecular signatures of disease for diagnosis and the prediction of a patient's response to treatment. These signatures will be composed of biomarkers specific to the disease, and will include over-expression of normal protein from a gene that does not carry a mutation; loss of expression of an essential protein; expression of a protein from a mutant gene; and metabolites whose levels are altered in disease. Surrogates for protein expression, such as alterations in the messenger RNA that encode for them, have already proved their value. The next challenge, then, in clinical biosensing is to enable the multiplexed detection of protein biomarkers, and perhaps the multiplexed detection of mixed biomarkers (metabolites, RNA and proteins) all in a single test. Given the plethora of available antibodies specific for biomarkers, why is this not already happening? We believe that the limitation lies in the nature of the antibody molecule itself, and especially the fact that antibodies have evolved to function in solution, while most diagnostic tests take place at a surface. We have accordingly turned to the design of alternative antibodies, and have identified a protein that appears to be unusually stable on surfaces. The new, non-antibody, scaffold protein is derived from human Stefin A, a natural inhibitor of the cathepsin family of proteases. We have engineered this protein so that it lacks natural binding partners, and introduced a series of new binding surfaces through randomisation or directed replacement of the surfaces used by Stefin A to bind to cathepsins. Our new probes show exquisite specificity and binding affinities comparable to antibodies, and can be used to probe biology in intact cells. More importantly, together and in collaboration with other groups in Chemistry or Engineering Departments, we have shown that these designer proteins can be used in optical detection of labelled target proteins from whole cell lysates in a highly multiplexed microarray format, as well as in label-free detection of unlabelled proteins using surface plasmon resonance, QCM, microcantilevers and using electrochemical assays on gold electrodes. We believe that the combination of optimised surface chemistry, robust and combinatorial designer biological probes and novel, robust and sensitive detection technologies will enable, in the near future, the introduction of multiplexed biomarker detection in the clinical setting, most likely in cancer where multiple biomarkers are known, but probes are still lacking.

Purified and Oxidized Single-Walled Carbon Nanotubes as Robust Near-IR Fluorescent Probes for Molecular Imaging

Single-walled carbon nanotubes (SWNTs) are suitable for many applications in molecular imaging because of their intrinsic near-infrared (NIR) photoluminescence. Nevertheless, several important issues, such as the SWNTs low biocompatibility, still need to be overcome for the effective use of carbon nanotubes in molecular imaging. In this study, we employ a two-step oxidation procedure with the aims of (1) removing the toxic metal impurities, (2) decreasing the lengths of the tubes, and (3) introducing functional oxygenated groups on the SWNT surface to enable the future development of a multifunctional SWNT-based nanoplatform. Since the presence of defect sites on the SWNT surface is known to quench the optical properties of carbon nanotubes, we have established what effect these two chemical treatments have on the NIR photoluminescence (PL) of the tubes. Our results show that purified and oxidized SWNT samples still display structured emission features in the NIR when dispersed in surfactant aqueous solution, with a residual emission efficiency of 30%. Two-dimensional (2D) fluorescence lifetime imaging (FLIM) of aggregates of purified and oxidized SWNTs in dimethylformamide (DMF) showed an average lifetime of 1.3 and 1.2 ns, respectively. These results demonstrate the relevance of SWNT purification and oxidation in the development of novel NIR probes with reduced toxicological impact on human health and with potential for further functionalization with multiple bioactive species.

Optimally designed nanolayered metal-dielectric particles as probes for massively multiplexed and ultrasensitive molecular assays

An outstanding challenge in biomedical sciences is to devise a palette of molecular probes that can enable simultaneous and quantitative imaging of tens to hundreds of species down to ultralow concentrations. Addressing this need using surface-enhanced Raman scattering-based probes is potentially possible. Here, we theorize a rational design and optimization strategy to obtain reproducible probes using nanospheres with alternating metal and reporter-filled dielectric layers. The isolation of reporter molecules from metal surfaces suppresses chemical enhancement, and consequently signal enhancements are determined by electromagnetic effects alone. This strategy synergistically couples interstitial surface plasmons and permits the use of almost any molecule as a reporter by eliminating the need for surface attachment. Genetic algorithms are employed to optimize the layer dimensions to provide controllable enhancements exceeding 11 orders of magnitude and of single molecule sensitivity for nonresonant and resonant reporters, respectively. The strategy also provides several other opportunities, including a facile route to tuning the response of these structures to be spectrally flat and localization of the enhancement within a specific volume inside or outside the probe. The spectrally uniform enhancement for multiple excitation wavelengths and for different shifts enables generalized probes, whereas enhancement tuning permits a large dynamic range by suppression of enhancements from outside the probe. Combined, these theoretical calculations open the door for a set of reproducible and robust probes with controlled sensitivity for molecular sensing over a concentration range of over 20 orders of magnitude.

Development of a Portable Probe for Field and Laboratory Measurement of Low to Medium Values of Soil Suction

Abstract The sample quality suction probe is an economical, robust, and portable probe capable of obtaining rapid and reliable, direct, non-destructive measurements of soil suction in the field or laboratory. Based on the designs of Ridley and Burland in 1993 and Toker et al. in 2004 for unsaturated soils, this probe is constructed mainly from commercially available components and the saturation and measurement procedures are easy to perform. The probe is able to obtain repeatable measurements from saturated soil samples in the field immediately after sampling and also in the laboratory prior to specimen preparation for consolidation or strength testing. These suction measurements may be used to evaluate sample quality in the field or in the laboratory prior to specimen testing.

Online Process Control of a Pharmaceutical Intermediate in a Fluidized-Bed Drier Environment Using Near-Infrared Spectroscopy

In the production plant of an antibiotic substance, a new fluidized-bed drier has been installed. For online process control of the drying progress and determination of the ideal drying end point, a continuous near-infrared spectroscopic (NIRS) measuring setup was implemented to rapidly and simultaneously gain all essential product information. A bypass system outside the drier combined with a robust process probe proved to provide the best sampling system geometry. The spectrometer was equipped with an additional laboratory probe for complementary offline analysis. Multivariate calibrations for product assay, water content, and residual solvent were calculated, optimized, and compared for the two probes. The final root-mean-square error of cross validation (RMSECV) for the process probe could be reduced to 0.81% for the product assay, 0.25% for water, and 0.06% for acetone. The laboratory-probe prediction values show good agreement with reference data during the testing period. The calibrations of the process probe were checked by comparing its predictions to those of the validated laboratory probe. The monitoring system could be automated to a large extent, and product quality could be improved considerably. The established technology is of high importance for the pharmaceutical industry carrying out high-throughput routine analysis because of its advantages in terms of of time and cost reductions.

Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL® vaccine-relevant HPV types (6, 11, 16 and 18). These probes are obtained through i<em>n vitro </em>selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5+/6+ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109) and human DNA concentrations (10-100 ng) as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL® vaccine-relevant HPV types (6, 11, 16 and 18). These probes are obtained through in vitro selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5+/6+ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109) and human DNA concentrations (10-100 ng) as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

Simple kinetic sensor to structural transitions

Driven nonequilibrium structural phase transformation has been probed using time-varying resistance fluctuations or noise. We demonstrate that the non-Gaussian component (NGC) of noise obtained by evaluating the higher-order statistics of fluctuations, serves as a simple kinetic detector of these phase transitions. Using the Martensite transformation in free-standing wires of nickel-titanium binary alloys as a prototype, we observe clear deviations from the Gaussian background in the transformation zone, indicative of the long-range correlations in the system as the phase transforms. The viability of non-Gaussian statistics as a robust probe to structural phase transition was also confirmed by comparing the results from differential scanning calorimetry measurements. We further studied the response of the NGC to the modifications in the microstructure on repeated thermal cycling, as well as the variations in the temperature-drive rate, and explained the results using established simplistic models based on the different competing time scales. Our experiments (i) suggest an alternative method to estimate the transformation temperature scales with high accuracy and (ii) establish a connection between the material-specific evolution of microstructure to the statistics of its linear response. Since the method depends on an in-built long-range correlation during transformation, it could be portable to other structural transitions, as well as to materials of different physical origin and size.