Detection of protein biomarkers using RNA aptamer microarrays and enzymatically amplified surface plasmon resonance imaging

A methodology for the detection of protein biomarkers at picomolar concentrations that utilizes surface plasmon resonance imaging (SPRI) measurements of RNA aptamer microarrays is developed. The adsorption of proteins onto the RNA microarray is detected by the formation of a surface aptamer-protein-antibody complex. The SPRI response signal is then amplified using a localized precipitation reaction catalyzed by the enzyme horseradish peroxidase that is conjugated to the antibody. This enzymatically amplified SPRI methodology is first characterized by the detection of human thrombin at a concentration of 500 fM; the appropriate thrombin aptamer for the sandwich assay is identified from a microarray of three potential thrombin aptamer candidates. The SPRI method is then used to detect the protein vascular endothelial growth factor (VEGF) at a biologically relevant concentration of 1 pM. VEGF is a signaling protein that has been used as a serum biomarker for rheumatoid arthritis, breast cancer, lung cancer, and colorectal cancer and is also associated with age-related macular degeneration.     

Real-time volume imaging using a crossed electrode array

This paper describes a unique crossed electrode array for real-time volume ultrasound imaging. By placing orthogonal linear array electrode patterns on the opposite sides of a hemispherically shaped composite transducer substrate, a 2D array can be fabricated using a small fraction of the elements required for a traditional 2D array. The performance of the array is investigated using a computer simulation of the radiation pattern. We show that by using a 288-element crossed electrode pattern it is possible to collect large field of view volume images (60deg times 60degsector) at real-time frame rates (>20 volume images/s), with image contrast and resolution comparable to what can be obtained using a conventional 128-element linear phased array.     

Real-time 3-D ultrasound imaging using sparse synthetic aperture beamforming

A method for real-time three-dimensional (3-D) ultrasound imaging using a mechanically scanned linear phased array is proposed. The high frame rate necessary for real-time volumetric imaging is achieved using a sparse synthetic aperture beamforming technique utilizing only a few transmit pulses for each image. Grating lobes in the two-way radiation pattern are avoided by adjusting the transmit element spacing and the receive aperture functions to account for the missing transmit elements. The signal loss associated with fewer transmit pulses is minimized by increasing the power delivered to each transmit element and by using multiple transmit elements for each transmit pulse. By mechanically rocking the array, in a way similar to what is done with an annular array, a 3-D set of images can be collected in the time normally required for a single image.     

Real-time rectilinear volumetric imaging

Current real-time volumetric scanners use a 2-D array to scan a pyramidal volume consisting of many sector scans stacked in the elevation direction. This scan format is primarily useful for cardiac imaging to avoid interference from the ribs. However, a real-time rectilinear volumetric scan with a wider field of view close to the transducer could prove more useful for abdominal, breast, or vascular imaging. In previous work, computer simulations of very sparse array transducer designs in a rectilinear volumetric scanner demonstrated that a Mills cross array showed the best overall performance given current system constraints. Consequently, a 94×94 Mills cross array including 372 active channels operating at 5 MHz has been developed on a flexible circuit interconnect. In addition, the beam former delay software and scan converter display software of the Duke volumetric scanner were modified to achieve real-time rectilinear volumetric scanning consisting of a 30-mm×8-mm×60-mm scan at a rate of 47 volumes/s. Real-time rectilinear volumetric images were obtained of tissue-mimicking phantoms, showing a spatial resolution of 1 to 2 mm. Images of carotid arteries in normal subjects demonstrated tissue penetration to 6 cm     

Enzyme-linked small-molecule detection using split aptamer ligation

Here we report an aptamer-based analogue of the widely used sandwich enzyme-linked immunosorbent assay (ELISA). This assay utilizes the cocaine split aptamer, which is comprised of two DNA strands that only assemble in the presence of the target small molecule. One split aptamer fragment is immobilized on a microplate, then a test sample is added containing the second split aptamer fragment. If cocaine is present in the test sample, it directs assembly of the split aptamer and promotes a chemical ligation between azide and cyclooctyne functional groups appended to the termini of the split aptamer fragments. Ligation results in covalent attachment of biotin to the microplate and provides a colorimetric output upon conjugation to streptavidin-horseradish peroxidase. Using this assay, we demonstrate detection of cocaine at concentrations of 100 nM-100 μM in buffer and 1-100 μM human blood serum. The detection limit of 1 μM in serum represents an improvement of two orders of magnitude over previously reported split aptamer-based sensors and highlights the utility of covalently trapping split aptamer assembly events.     

Real-time single-molecule imaging of quantum interference

The observation of interference patterns in double-slit experiments with massive particles is generally regarded as the ultimate demonstration of the quantum nature of these objects. Such matter-wave interference has been observed for electrons, neutrons, atoms and molecules and, in contrast to classical physics, quantum interference can be observed when single particles arrive at the detector one by one. The build-up of such patterns in experiments with electrons has been described as the "most beautiful experiment in physics". Here, we show how a combination of nanofabrication and nano-imaging allows us to record the full two-dimensional build-up of quantum interference patterns in real time for phthalocyanine molecules and for derivatives of phthalocyanine molecules, which have masses of 514?AMU and 1,298?AMU respectively. A laser-controlled micro-evaporation source was used to produce a beam of molecules with the required intensity and coherence, and the gratings were machined in 10-nm-thick silicon nitride membranes to reduce the effect of van der Waals forces. Wide-field fluorescence microscopy detected the position of each molecule with an accuracy of 10?nm and revealed the build-up of a deterministic ensemble interference pattern from single molecules that arrived stochastically at the detector. In addition to providing this particularly clear demonstration of wave-particle duality, our approach could also be used to study larger molecules and explore the boundary between quantum and classical physics.     

Real-time adaptive clutter rejection filtering in color flow imaging using power method iterations

We propose a new algorithm for real-time, adaptive-clutter-rejection filtering in ultrasound color flow imaging (CFI) and related techniques. The algorithm is based on regression filtering using eigenvectors of the signal correlation matrix as a basis for representing clutter, a method that previously has been considered too computationally demanding for real-time processing in general CFI applications. The data acquisition and processing scheme introduced allows for a more localized sampling of the clutter statistics and, therefore, an improved clutter attenuation for lower filter orders. By using the iterative power method technique, the dominant eigenvalues and corresponding eigenvectors of the correlation matrix can be estimated efficiently, rendering real-time operation feasible on desktop computers. A new adaptive filter order algorithm is proposed that successfully estimates the proper dimension of the clutter basis, previously one of the major drawbacks of this clutter-rejection technique. The filter algorithm performance and computational demands has been compared to that of conventional clutter filters. Examples have been included which confirms that, by adapting the clutter-rejection filter to estimates of the clutter-signal statistics, improved attenuation of the clutter signal can be achieved in normal as well as more excessive cases of tissue movement and acceleration.     

Real-time ultrasound process tomography for two-phase flow imaging using a reduced number of transducers

In this paper a novel ultrasound tomography imaging system is presented. It employs a relatively small number of transducers that produce fan-shaped beam profile to effectively insonify the cross-section. The impact on the image quality due to the reduction of the number of transducers is discussed, and different approaches such as multiple receiver data acquisition and nonlinear thresholding are explored. A prototype of a tomographic imaging system with only 36 transducers has been constructed, and processing rates of up to 100 frames per second have been achieved using a parallel processing technique. Good image reconstructions based on simulations and real objects also are provided to confirm the principles of the theoretical analysis.     

Real-time protein kinase assay

We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.     

Near infrared surface plasmon resonance phase imaging and nanoparticle-enhanced surface plasmon resonance phase imaging for ultrasensitive protein and DNA biosensing with oligonucleotide and aptamer microarrays

The techniques of surface plasmon resonance-phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have been implemented for the multiplexed bioaffinity detection of proteins and nucleic acids. The SPR-PI experiments utilized a near-infrared 860 nm light emitting diode (LED) light source and a wedge depolarizer to create a phase grating on a four-element single-stranded DNA (ssDNA) microarray; bioaffinity adsorption onto the various microarray elements was detected via multiplexed real time phase shift measurements. In a first set of demonstration experiments, an ssDNA aptamer microarray was used to directly detect thrombin at concentrations down to 100 pM with SPR-PI. Two different ssDNA aptamers were used in these experiments with two different Langmuir adsorption coefficients, K(A1) = 4.4 × 10(8) M(-1) and K(A2) = 1.2 × 10(8) M(-1). At concentrations below 1 nM, the equilibrium phase shifts observed upon thrombin adsorption vary linearly with concentration with a slope that is proportional to the appropriate Langmuir adsorption coefficient. The observed detection limit of 100 pM is approximately 20 times more sensitive than that observed previously with SPRI. In a second set of experiments, two short ssDNA oligonucleotides (38mers) were simultaneously detected at concentrations down to 25 fM using a three-sequence hybridization format that employed 120 nm DNA-modified silica nanoparticles to enhance the SPR-PI signal. In this first demonstration of nanoparticle-enhanced SPR-PI, the adsorbed silica nanoparticles provided a greatly enhanced phase shift upon bioaffinity adsorption due to a large increase in the real component of the interfacial refractive index from the adsorbed nanoparticle. As in the case of SPR-PI, the detection limit of 25 fM for nanoparticle-enhanced SPR-PI is approximately 20 times more sensitive than that observed previously with nanoparticle-enhanced SPRI.     

亚表面缺陷的实时成像检测

提出一种适合于金属亚表面缺陷的可视化无损检测方法——磁光/脉冲涡流成像方法。该方法以脉冲信号激励产生涡流,以激光对被检测物体的照射取代传统涡流检测的线圈探头,通过磁光传感元件将缺陷引起的磁场变化转换成相应的光强度的变化,由传统的显微镜、照明系统、偏振器和CCD图像传感器组成的光学系统将光强变化转换为“明”或“暗”图像,实现了对缺陷的实时成像检测。本文论述了磁光/脉冲涡流实时成像检测机理,给出了一种实验装置。通过对金属表面/亚表面缺陷实验,表明该检测方法快速、准确,可实现微/纳米级缺陷的成像检测。     

Real-time all-optical three-dimensional integral imaging projector

We present an all-optical three-dimensional integral imaging projector. An optically addressed spatial-light modulator is used, which potentially provides better image resolution than the conventional CCD and liquid-crystal display pair. We present experimental results using a liquid-crystal light valve.     

Identifying cell-to-cell variability in internalization using flow cytometry

Biological heterogeneity is a primary contributor to the variation observed in experiments that probe dynamical processes, such as the internalization of material by cells. Given that internalization is a critical process by which many therapeutics and viruses reach their intracellular site of action, quantifying cell-to-cell variability in internalization is of high biological interest. Yet, it is common for studies of internalization to neglect cell-to-cell variability. We develop a simple mathematical model of internalization that captures the dynamical behaviour, cell-to-cell variation, and extrinsic noise introduced by flow cytometry. We calibrate our model through a novel distribution-matching approximate Bayesian computation algorithm to flow cytometry data of internalization of anti-transferrin receptor antibody in a human B-cell lymphoblastoid cell line. This approach provides information relating to the region of the parameter space, and consequentially the nature of cell-to-cell variability, that produces model realizations consistent with the experimental data. Given that our approach is agnostic to sample size and signal-to-noise ratio, our modelling framework is broadly applicable to identify biological variability in single-cell data from internalization assays and similar experiments that probe cellular dynamical processes.     

Real-time extended dynamic range imaging in shearography

Extended dynamic range (EDR) imaging is a postprocessing technique commonly associated with photography. Multiple images of a scene are recorded by the camera using different shutter settings and are merged into a single higher dynamic range image. Speckle interferometry and holography techniques require a well-modulated intensity signal to extract the phase information, and of these techniques shearography is most sensitive to different object surface reflectivities as it uses self-referencing from a sheared image. In this paper the authors demonstrate real-time EDR imaging in shearography and present experimental results from a difficult surface reflectivity sample: a wooden panel painting containing gold and dark earth color paint.     

Real-time quantification of microscale bioadhesion events in situ using imaging surface plasmon resonance (iSPR)

From macro- to nanoscales, adhesion phenomena are all-pervasive in nature yet remain poorly understood. In recent years, studies of biological adhesion mechanisms, terrestrial and marine, have provided inspiration for "biomimetic" adhesion strategies and important insights for the development of fouling-resistant materials. Although the focus of most contemporary bioadhesion research is on large organisms such as marine mussels, insects and geckos, adhesion events on the micro/nanoscale are critical to our understanding of important underlying mechanisms. Observing and quantifying adhesion at this scale is particularly relevant for the development of biomedical implants and in the prevention of marine biofouling. However, such characterization has so far been restricted by insufficient quantities of material for biochemical analysis and the limitations of contemporary imaging techniques. Here, we introduce a recently developed optical method that allows precise determination of adhesive deposition by microscale organisms in situ and in real time; a capability not before demonstrated. In this extended study we used the cypris larvae of barnacles and a combination of conventional and imaging surface plasmon resonance techniques to observe and quantify adhesive deposition onto a range of model surfaces (CH(3)-, COOH-, NH(3)-, and mPEG-terminated SAMs and a PEGMA/HEMA hydrogel). We then correlated this deposition to passive adsorption of a putatively adhesive protein from barnacles. In this way, we were able to rank surfaces in order of effectiveness for preventing barnacle cyprid exploration and demonstrate the importance of observing the natural process of adhesion, rather than predicting surface effects from a model system. As well as contributing fundamentally to the knowledge on the adhesion and adhesives of barnacle larvae, a potential target for future biomimetic glues, this method also provides a versatile technique for laboratory testing of fouling-resistant chemistries.     

Signaling aptamer/protein binding by a molecular light switch complex

A novel method of signaling aptamer/protein binding for aptamer-based protein detection has been developed using a molecular light switch complex, [Ru(phen)2(dppz)]2+. The method takes advantage of the sensitive luminescence signal change of [Ru(phen)2(dppz)]2+ intercalating to the aptamer upon protein/aptamer binding. A 37-nt DNA aptamer against immunoglobulin E (IgE) was first tested as a model system. The luminescence of the [Ru(phen)2(dppz)]2+/IgE aptamer decreased with the increase of IgE. By monitoring the luminescence change, we were able to detect the binding events between the aptamer and IgE for IgE quantitation in homogeneous solutions as well as in serum. The assay was highly selective and sensitive with a detection limit of 100 pM for IgE. This new method is very simple and without the need for the covalent coupling of fluorophores to aptamers. The generalizability of the method was demonstrated by the direct detection of two other proteins, oncoprotein platelet derived growth factor-BB (PDGF-BB) using its DNA aptamer and alpha-thrombin using its RNA aptamer. This new approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.     

Ultrasensitive DNA detection using oligonucleotide-silver nanoparticle conjugates

Oligonucleotide-gold nanoparticle (OGN) conjugates are powerful tools for the detection of target DNA sequences due to the unique properties conferred upon the oligonucleotide by the nanoparticle. Practically all the research and applications of these conjugates have used gold nanoparticles to the exclusion of other noble metal nanoparticles. Here we report the synthesis of oligonucleotide-silver nanoparticle (OSN) conjugates and demonstrate their use in a sandwich assay format. The OSN conjugates have practically identical properties to their gold analogues and due to their vastly greater extinction coefficient both visual and absorption analyses can occur at much lower concentrations. This is the first report of OSN conjugates being successfully used for target DNA detection and offers improved sensitivity which is of interest to a range of scientists.     

MR imaging of Her-2/neu protein using magnetic nanoparticles

The aim of this study was to assess whether Her-2/neu expressing tumour cells can be detected in vitro as well as in animal tumour models with magnetic resonance imaging at 1.5?T. Magnetic nanoparticles (with relaxivities R 1, R 2 of 3.7 ± 0.4?(mM?s)(-1), 277 ± 32?(mM?s)(-1) at 21?°C, respectively) coupled to anti-Her-2/neu antibodies or gamma globulin IgG (high or non-affinity probe, respectively) were used. After incubation of Her-2/neu expressing cells (SKBR3) with high or non-affinity probes (20?min), values of R 1 = 0.34 ± 0.02?(mM?s)(-1) and R 2 = 63.02 ± 30?(mM?s)(-1) were obtained. Electron microscopy and atomic absorption spectrometry examinations verified the presence of relatively high iron levels in cells incubated with the high affinity probe compared to controls. For in vivo MRI, high or non-affinity probes (≈1.7?mg Fe/animal) were injected into the tail vein of mice (n = 16) bearing SKBR3 tumours. A distinct decrease in the normalized MR signal ratio between tumour and reference area (approximately -17 ± 2%) after application of the high affinity probe was observed. In conclusion, in vivo detection of Her-2/neu expressing tumours is feasible in a clinical MR scanner by using immunoconjugated magnetic nanoparticles.     

Fluorescence anisotropy reduction of allosteric aptamer for sensitive and specific protein signaling

Real time protein signaling in a complex medium may provide a promising way for high-throughput protein analysis, but it is largely unmet due to the challenge of signal transduction and the interferences of nonspecific binding and high background. Our recent work indicates that a fluorescent aptamer can display a protein binding-induced reduction of fluorescence anisotropy (FA) (Zhang, D.; Lu, M.; Wang, H. J. Am. Chem. Soc. 2011, 133, 9188-9191), which is exclusively different from a traditionally simplified concept hinting a molecular size increase-induced FA increase. Inspired by this unexpected observation, we describe a novel FA reduction approach for protein signaling. The feasibility of this approach is demonstrated through the assays of a blood protein human α-thrombin and an oncoprotein human platelet-derived growth factor B-chain (PDGF-BB) using two screened fluorescent aptamers, respectively. By the developed FA reduction method, the spiked human α-thrombin in diluted serum can be detected at the concentration as low as 250 pM. In contrast, in a traditional molecular size-dependent FA assay, the thrombin spiked in diluted serum cannot induce reliable FA change even at a 256-fold higher concentration (64 nM). The results clearly show that the FA reduction approach has a dramatically enhanced specificity against target protein and high sensitivity in complex medium and is applicable to the no-separation based detection of proteins in biological matrixes.