Diagnostic Array Research Articles

Influence of Dangling Ends and Surface-Proximal Tails of Targets on Probe-Target Duplex Formation in 16S rRNA Gene-Based Diagnostic Arrays

Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.

Quantitative Analysis of Tethered Vesicle Assemblies by Quartz Crystal Microbalance with Dissipation Monitoring: Binding Dynamics and Bound Water Content

To implement the molecular recognition properties of membrane proteins for applications including biosensors and diagnostic arrays, the construction of a biomimetic platform capable of maintaining protein structure and function is required. In this paper, we describe a tethered phospholipid vesicle assembly that overcomes the major limitations of planar supported lipid bilayers and alternative biomimetic membrane platforms and characterize it using quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy. We provide evidence of a one-step mechanism for bilayer formation and monitor the subsequent adsorption and binding of streptavidin, vesicles, and streptavidin-coated microspheres. For all three species, we identify a critical surface density above which a significant amount of coupled interstitial water contributes to the response of the quartz resonator in a phenomenon similar to dynamic coupling due to surface roughness. A Sauerbrey-type analysis is sufficient to accurately interpret the QCM-D results for streptavidin binding if water is treated as an additional inertial mass, but viscoelastic models must be invoked for vesicle and microsphere binding. Additionally, we present evidence of vesicle flattening, possibly enhanced by a biotin-mediated membrane-membrane interaction.

Development of a membrane array‐based multimarker assay for detection of circulating cancer cells in patients with non‐small cell lung cancer

Combination of multiple mRNA markers has been largely investigated for detection of circulating cancer cells. However, current PCR-based methods are relatively expensive and time consuming. The aim of this study was to develop a membrane array-based multimarker assay for detection of circulating cancer cells in nonsmall cell lung cancer (NSCLC) patients. At first, we selected 22 candidate genes by means of suppression subtractive hybridization and Northern blot analysis. The diagnostic value of each candidate gene was then preliminarily evaluated in 50 pairs of blood samples by membrane array method. Accordingly, 17 genes with area under the ROC curve (AUC) > or = 0.8 were selected as target genes to reconstruct the diagnostic membrane array, which was then used to test peripheral blood samples from 100 NSCLC patients and 147 control subjects. ROC curve analysis demonstrated that the optimal threshold number of overexpressed markers on membrane array for discrimination between NSCLC patients and control subjects was 12. As a result, the diagnostic membrane array could detect circulating cancer cells in 90 (90%) of 100 NSCLC patients and in 14 (9.5%) of 147 control subjects (including 6 of 100 normal persons, 3 of 20 breast cancer patients, 3 of 15 colorectal cancer patients and 2 of 12 gastric cancer patients). Moreover, the detection rate was significantly correlated with NSCLC patients' metastatic status and overall stage (p = 0.028 and 0.014, respectively). These results suggested that our blood-based membrane array assay for molecular detection of circulating lung cancer cells has great potential for clinical applications.

Specialized DNA Arrays for the Differentiation of Pancreatic Tumors

Malignant tumors of the pancreas are frequently indistinguishable from inflammatory tumors arising in the context of a chronic pancreatitis with the use of conventional imaging techniques. Thus, cytologic analysis of cells obtained by abdominal ultrasound, computed tomography, or endoscopic ultrasound-guided fine needle aspiration biopsy is required for diagnosis. However, the reliability of cytologic analyses of pancreatic fine needle aspirates remains unsatisfactory, with a diagnostic accuracy of < or =80%. The purpose of the current study was therefore to develop a novel diagnostic approach based on expression profiling of biopsy material using a specialized diagnostic cDNA array. Previous gene expression profiling studies were reevaluated to design a 558-feature diagnostic array. Minimal amounts of residual material from pancreatic cytology samples as well as surgically resected tumor and control tissue specimens were analyzed using the diagnostic array and a newly developed statistical classification system. Our diagnostic approach resulted in 95% accurate differentiation between ductal adenocarcinomas and nonmalignant tumors of the pancreas. The diagnostic array, in conjunction with conventional diagnostic procedures, is thus suitable to significantly improve the reliability of pancreatic cancer diagnostics and can be expected to become a valuable new tool in the routine workup of suspect masses in the pancreas.

X-ray flux from filtered arrays of detectors without unfolding

A simple computational method is proposed for estimating the time-dependent flux F[ΔE](t) of an x-ray spectrum S(E,t) over domain [ΔE] from data Dk(t)(k=1,…,N) obtained by an N-channel array of filtered detectors. It is assumed that the data are related to the spectrum by a discrete, inhomogeneous, first-kind Fredholm integral equation Dk=∫S(E,t)Rk(E)dE, where Rk(E) is the known response function for each detector channel of the diagnostic. The proposed method constructs a spectral sensitivity HLS(E) for the diagnostic array as a linear combination ∑k=1NakRk(E) of the responses, where the coefficients ak are obtained by a least-squares criterion plus a constraint. The ak values, once determined, apply as long as the responses are valid. The flux estimate is then simply FLS(t)=∑k=1NakDk(t), without a spectral unfold of the data. The method is useful for quick analyses of time-dependent data, for comparisons with other flux-measuring diagnostics, and for the experimental design of filtered-detector arrays. The method is applied to a five-channel array of filtered photoemissive x-ray detectors [G. A. Chandler et al., Rev. Sci. Instrum. 70, 561 (1999)], used for z-pinch measurements at the Z-accelerator facility [R. B. Spielman et al., Phys. Plasmas 5, 2105 (1998)]. Comparisons with unfold results are made, and a first-order analysis of error propagation into FLS(t) is presented.

High spatial resolution Hall sensor array for edge plasma magnetic field measurements

A one-dimensional, high-spatial resolution, 20-element Hall sensor array has been developed to directly measure the edge plasma perpendicular magnetic field and its fluctuations as a function of radius with 4-mm resolution. The array employs new small-area, high-sensitivity indium antimonide (InSb) Hall probes in combination with a high-density seven-layer printed circuit board to provide for connections to supply Hall current, record the measured Hall voltage output signals, and mitigate inductive pickup. A combination of bench and in situ measurements is described that provides absolute calibration of the diagnostic array in the presence of a strong transverse magnetic field component that is approximately 1000 times greater than the perpendicular fluctuating field needed to be resolved by the diagnostic. The Hall probes calibrated using this method are capable of magnetic field measurements with a sensitivity of 7V∕T over the frequency band from 0 to 20 kHz.

Detection of circulating cancer cells with K-ras oncogene using membrane array

K-ras oncogene is frequently found in human cancers and thus may serve as a potential diagnostic marker for cancer cells in circulation. So far, there is no reliable method for detecting cancer cells with K-ras oncogene in peripheral blood. The objective of this study was to develop a diagnostic membrane array using activated K-ras oncogene-associated molecules as detection targets. In our previous study, cDNA microarray analysis showed that there were 94 genes differentially expressed in K-ras mutant stably transfected adrenocortical cells. In the present study, we obtained 22 up-regulated genes in the closest relation to K-ras oncogene through bioinformatic analysis. At first, we carried out membrane array analysis by using in vitro culture cells. We demonstrated that this diagnostic technique was feasible and highly sensitive. A number as low as 5 cancer cells bearing K-ras oncogene in 1 ml of blood could be distinctively detected. Then, we collected blood specimens from 76 cancer patients. Direct sequencing analysis of these 76 samples showed that K-ras mutation was present in 43 patients with mutation sites mainly at codons 13, 15 and 61, which have been commonly established to be activated sites. We subsequently analyzed these 76 specimens with our diagnostic membrane array. Thirty-nine specimens were detected as positive for activated K-ras oncogene. Eighty percent (12/15) of mutations occurred at codon 13, 72.7% (8/11) at codon 61, and 88.9% (8/9) at codon 15 were accurately detected by our diagnostic membrane. Finally, through a series of biostatistical analyses, the sensitivity, specificity and accuracy of the diagnostic membrane array were 83.7, 90.9 and 86.8%, respectively. These findings suggest that the K-ras oncogene membrane array has a great potential for further investigation and clinical application.

Hybridization isotherms of DNA microarrays and the quantification of mutation studies.

Diagnostic DNA arrays for detection of point mutations as markers for cancer usually function in the presence of a large excess of wild-type DNA. This excess can give rise to false positives as a result of competitive hybridization of the wild-type target at the mutation spot. Analysis of the DNA array data is typically qualitative, aimed at establishing the presence or absence of a particular point mutation. Our theoretical approach yields methods for quantifying the analysis to obtain the ratio of concentrations of mutated and wild-type DNA. The theory is formulated in terms of the hybridization isotherms relating the hybridization fraction at the spot to the composition of the sample solutions at thermodynamic equilibrium. It focuses on samples containing an excess of single-stranded DNA and on DNA arrays with a low surface density of probes. The hybridization equilibrium constants can be obtained by the nearest-neighbor method. Two approaches allow acquisition of quantitative results from the DNA array data. In one, the signal of the mutation spot is compared with that of the wild-type spot. The implementation requires knowledge of the saturation intensity of the two spots. The second approach requires comparison of the intensity of the mutation spot at two different temperatures. In this case, knowledge of the saturation signal is not always necessary. DNA arrays can be used to obtain quantitative results on the concentration ratio of mutated DNA to wild-type DNA in studies of somatic point mutations.

Use of an oligonucleotide array for laboratory diagnosis of bacteria responsible for acute upper respiratory infections.

We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.

Design and testing of an ultrasonic system with integrated diagnostic and therapy modes

Our laboratories developed and tested a system integrating high-intensity focused ultrasound (HIFU) with diagnostic ultrasound procedures for aiming, delivering, and monitoring treatment exposures. The system employs 5-MHz, 5-element spherical-cap HIFU arrays with annular electrodes (for variable focusing) or strip electrodes (for inducing broad lesions of controlled width). Central apertures in these arrays house a coaxial diagnostic phased array (48 elements), interfaced with a custom digital system for visualization and RF echo acquisition. Several operational modes were investigated. A preexposure mode permits the HIFU focal point to be aimed at the desired location in B-mode images. It also assesses harmonic generation in the HIFU beam, using the diagnostic array to receive and spectrally analyze echoes from a short HIFU pulse aimed at targeted tissues. Radiation-force elastography uses the diagnostic array to monitor tissue displacements induced by brief exposures from the HIFU transducer; pre- and posttherapy data are compared to delineate mechanical alterations in lesioned volumes. Acquired pre- and post-RF data are also spectrally analyzed to map changes in scattering and attenuation indicative of lesion production. These modes have been successfully tested under in vitro conditions and are now entering use in animal studies aimed at treating cancer and cardiac diseases.

DNA microarray technology in nutraceutical and food safety

The quality and quantity of diet is a key determinant of health and disease. Molecular diagnostics may play a key role in food safety related to genetically modified foods, food-borne pathogens and novel nutraceuticals. Functional outcomes in biology are determined, for the most part, by net balance between sets of genes related to the specific outcome in question. The DNA microarray technology offers a new dimension of strength in molecular diagnostics by permitting the simultaneous analysis of large sets of genes. Automation of assay and novel bioinformatics tools make DNA microarrays a robust technology for diagnostics. Since its development a few years ago, this technology has been used for the applications of toxicogenomics, pharmacogenomics, cell biology, and clinical investigations addressing the prevention and intervention of diseases. Optimization of this technology to specifically address food safety is a vast resource that remains to be mined. Efforts to develop diagnostic custom arrays and simplified bioinformatics tools for field use are warranted.

Microarray Data Analysis: From Hypotheses to Conclusions Using Gene Expression Data

We review several commonly used methods for the design and analysis of microarray data. To begin with, some experimental design issues are addressed. Several approaches for pre‐processing the data (filtering and normalization) before the statistical analysis stage are then discussed. A common first step in this type of analysis is gene selection based on statistical testing. Two approaches, permutation and model‐based methods are explained and we emphasize the need to correct for multiple testing. Moreover, powerful approaches based on gene sets are mentioned. Clustering of either genes or samples is frequently performed when analyzing microarray data. We summarize the basics of both supervised and unsupervised clustering (classification). The latter may be of use for creating diagnostic arrays, for example. Construction of biological networks, such as pathways, is a statistically challenging but complex task that is a relatively new development and hence mentioned only briefly. We finish with some remarks on literature and software. The emphasis in this paper is on the philosophy behind several statistical issues and on a critical interpretation of microarray related analysis methods.

Ultrasonic diagnostic imaging system transducer array with multiline patches

An ultrasonic imaging method and apparatus are described for imaging the coronary arteries of the heart. The vascular system is infused with an ultrasonic contrast agent. A volumetric region of the heart wall including a coronary artery is three dimensionally scanned. A projection image of the volumetric region is produced from the scanning, providing a two dimensional contrast image of the coronary artery with the appearance of an angiogram. Preferably the coronary artery signals are segmented from contrast signals emanating from the myocardium and the heart blood pool so that the coronary arteries are clearly highlighted and distinct in the ultrasonic angiogram.

Teaching Decoding for Generalization Using the Nonverbal Reading Approach

The purpose of this study was to examine the effectiveness of teaching students with severe speech and physical impairments to decode words using the Nonverbal Reading Approach (NRA), and to determine if the students would generalize the decoding skills to unknown words with similar phoneme sequences. A multiple baseline probe design was used across 3 students. Results indicate that the NRA's use of internal speech, diagnostic distractor arrays, and error analysis was an effective means of teaching the students to decode targeted words. Use of these decoding skills generalized to unknown words with little or no additional instruction.

AlGaN UV Focal Plane Arrays

This paper presents characterization data, including UV imagery, for 256 × 256 AlGaN UV Focal Plane Arrays (FPAs). The UV-FPAs have 30 × 30 μm2 unit cells, and use back-illuminated arrays of AlGaN p–i–n photodiodes operating at zero bias voltage. The photodiode arrays were fabricated from multilayer AlGaN films grown by MOCVD on sapphire substrates. Data are also presented for individual AlGaN photodiodes and variable-area diagnostic arrays.

AlGaN UV Focal Plane Arrays

This paper presents characterization data, including UV imagery, for 256 x 256 AlGaN UV Focal Plane Arrays (FPAs). The UV-FPAs have 30 x 30 μm 2 unit cells, and use back-illuminated arrays of AlGaN p-i-n photodiodes operating at zero bias voltage. The photodiode arrays were fabricated from multilayer AlGaN films grown by MOCVD on sapphire substrates. Data are also presented for individual AlGaN photodiodes and variable-area diagnostic arrays.

The potential of liquid xenon for WIMP search: the ZEPLIN diagnostic array

A Liquid Xenon based WIMP detector diagnostic array is currently developed by the UKDMC with the help of international collaborators. After a brief reminder on the detection principle in Liquid Xenon, the individual detectors will be described. ZEPLIN I, a detector with a 4 kg fiducial mass with a background discrimination based on Pulse Shape Analysis, is already underground and starting operation. Two setups with improved background discrimination tools (as the ionisation is also recorded) are designed and scheduled to move underground in the second half of 2001. Both of them, ZEPLIN II and ZEPLIN III, are predicted to be sensitive to rate of 0.1–0.01 events/kg/day within 2 years of data taking. Furthermore, new ideas for lower background readout devices are studied, in order to avoid the use of PhotoMultiplier Tubes (PMTs).

Influence of beam geometry and tissue boundaries on thermal ultrasonic lesions

The size and shape of thermal lesions induced by high-intensity focused ultrasound (HIFU) are key factors in treating disease. Studies are being conducted to determine how HIFU beams and exposures can be designed to produce useful lesion attributes in specific target tissues. The studies include radially asymmetric beams and tissues with different acoustic absorption coefficients; sharp thermal gradients at such boundaries can significantly affect lesion geometry. Ocular tumor therapy uses asymmetric beams that produce lesions with ellipsoidal cross sections to facilitate treatments with closely packed lesion matrices. Three-dimensional simulations have analyzed heat patterns and lesions induced by these beams in irregular tumors whose geometry is derived from clinical serial-plane scans using 50-MHz diagnostic ultrasound. The simulations documented difficulties in treating boundary regions adjacent to the waterlike vitreous humor. Lesion results were consistent with in vitro and in vivo animal experiments. A related study involves an annular-array therapy system with a central rectangular aperture, housing a diagnostic array. The system (Spectrasonics, Inc.) is intended for treatments in various organs, including liver. Since the therapy beams are not radially symmetric, 3-D simulations and initial animal experiments have examined the asymmetry in induced temperature patterns and lesions. These investigations also addressed spurious grating peaks in axial intensity.

MOVPE growth of HgCdTe for high performance 3–5 µm photodiodes operating at 100–180K

New results are reported on the growth of high performance medium wavelength infrared (3–5 µm) (MWIR) HgCdTe photodiodes in the three-layer P-n-N configuration. The detector structures were grown in situ by metalorganic vapor phase epitaxy (MOVPE) on (211)B oriented CdZnTe substrates. The mobilities of the single n-type layers with x-values of ∼0.30 are in the range of (3–4.5)×104 cm2/V-s at 80K. The lifetimes on unpassivated films range from 1–5 and 4–10 µs at 80 and 180K, respectively, which are within a factor of two or less of the lifetimes calculated for Auger-1 and radiative recombination. The P-n-N films were processed into variable-area backside-illuminated diagnostic arrays and tested for quantum efficiency, spectral response, RDA, I–V curves and 1/f noise in the 120–180K range. The internal one-dimensional quantum efficiencies are in the range of 85–100%. The optical collection lengths are typically ∼25 µm. I–V curves showed that diffusion current is the dominant junction current mechanism for temperatures ≥100K. R0A values are at the one-dimensional limit for n-side diffusion currents over the 100–180K range. 1/f noise was measured to be very low at 120K and is the same as that measured in similarly processed arrays from recent LPE grown P-on-N heterojunctions. The results demonstrate that MOVPE growth can be used for large area, high performance MWIR HgCdTe detector arrays operating in the 120–180K temperature range.

Ultrasonic diagnostic transducer array with elevation focus

An ultrasonic diagnostic transducer array is provided for providing electronic focusing in the longitudinal plane and elevational focusing. Elements of the array are subdiced in the elevational direction to provide subelements with aspect ratios varying in proportion to their distance to the central longitudinal axis of the array. Such variation affords varying electro-mechanical coupling coefficients to the subelements such that the intensity of the transmitted energy is centered about the central longitudinal axis. In a second embodiment elements exhibit extensions in the elevational direction which vary in proportion to their displacement from the longitudinal center of the array. The extended elements are acoustically separated into subelements in the elevational direction to provide elevational focusing or spatial compounding of the transmitted acoustic energy.