High-performance real-time quantitative RT-PCR using lanthanide probes and a dual-temperature hybridization assay

We report a time-resolved fluorescence-based, homogeneous approach for multiplex, real-time or end-point detection of PCR products. Signal generation consists of PCR associated digestion of a 5'-labeled oligonucleotide probe, rapid cooling of the reaction mixture, and hybridization of undigested probe oligonucleotides with a complementary, shorter probe that incorporates a quencher at its 3' end. The signal coming from intact fluorescent probe molecules is, thus, quenched. The fluorophores we have used are environmentally sensitive lanthanide chelates. Their signals can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. Signal-to-noise ratios are further enhanced by the environmental sensitivity of these chelates; they exhibit a higher fluorescence intensity when free in solution than when coupled to intact probe molecules. Because of the minimal background fluorescence, the signal-to-noise ratios are higher and threshold cycles are lower than those obtained using conventional TaqMan probes. The multiplexing capacity of the assay chemistry is demonstrated through simultaneous amplification and detection of prostate specific antigen (PSA) cDNA and an internal standard mRNA (mmPSA) using probes labeled with terbium and europium. The applicability of the assay chemistry to routine clinical diagnostics is demonstrated through absolute quantification of PSA mRNA in peripheral blood.     

Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection

Large-scale investigations of sequence variation within the human species will provide information about the basis of heritable variation in disease susceptibility and human migration. The surface invader assay (an adaptation of the invasive cleavage reaction to an array format) is capable of exquisitely sensitive and specific detection of genetic variation. It is shown here that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of single nucleotide polymorphisms (SNPs) directly from an unamplified genomic DNA target. In addition, a "universal" mode of detection was developed that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high-density arrays able to analyze hundreds of thousands or even millions of SNPs.     

Quadruple-analyte chemiluminometric hybridization assay. Application to double quantitative competitive polymerase chain reaction

We developed a highly sensitive quadruple-analyte chemiluminometric hybridization assay for simultaneous quantification of four nucleic acid sequences. The targets are amplified by the polymerase chain reaction (PCR) and captured to microtiter wells coated with streptavidin. The immobilized fragments are hybridized with specific probes containing a sequence complementary to the target and a sequence or a hapten that allows linkage with a chemiluminescent reporter. We prepared a mixture of four reporters conjugated to complementary oligonucleotides or antihapten antibodies. The reporters were aequorin-(dT)(30), galactosidase-oligonucleotide, horseradish peroxidase-antifluorescein, and alkaline phosphatase-antidigoxigenin conjugates. The four chemiluminescent reactions were triggered sequentially. The signals were linearly related to the concentration of target sequences. The entire quadruple-analyte bioluminometric hybridization assay is complete in 75 min. We have demonstrated the applicability of the proposed assay to high-throughput quantitative competitive PCR of two target sequences in the presence of the corresponding competitors. The assay is universal since the same reporter conjugates can be used for multianalyte quantification of any sequences with properly designed probes.     

Molecular architectures for electrocatalytic amplification of oligonucleotide hybridization

In this work, we describe a new platform suitable for electrocatalytic amplification of oligonucleotide hybridization based on the use of supramolecular bioconjugates incorporating ferrocene-labeled streptavidin. Our goals were aimed at designing a biosensing platform which could support highly reproducible and stable electrocatalytic amplification with maximum efficiency. The use of nonlabeled streptavidin as an underlying layer promotes a major improvement on the characteristics of the amplified electrochemical signal. In addition, the electrocatalytic current can be easily amplified by tuning the concentration of electron donor species in solution. Because of the fact that the redox labels are bioconjugated to the DNA strands, increasing the ionic strength does not lead to the loss of redox labels. More importantly, increasing the concentration of donors only involves the magnification of the signal without implying the permeation of donors (thus reducing the efficient electrocatalysis). This approach represents a major improvement on the use of electrocatalytically amplified DNA-sensing platforms, thus overcoming any possible limitation in connection with the reproducibility and reliability of this well-established method.     

Multiplex quantitative competitive polymerase chain reaction based on a multianalyte hybridization assay performed on spectrally encoded microspheres

Quantitative polymerase chain reaction (PCR) may be performed by two general approaches, namely, real-time PCR and quantitative competitive PCR (QC-PCR). QC-PCR makes use of the concept of a DNA competitor, which is the "gold standard" approach to circumvent the problem of the variation of amplification efficiency. However, QC-PCR in its classical form is a low-throughput method since it requires titration of each sample with the competitor followed by electrophoresis. The throughput of QC-PCR has been improved by capillary electrophoresis and microtiter well-based hybridization assays. The present work introduces a multiplex QC-PCR method, which is based on a multianalyte hybridization assay that is performed on spectrally encoded microspheres. The DNA competitors use the same primers and have equal size with their corresponding target DNA sequences but differ in a short (24 bp) centrally located sequence. Following multiplex PCR, biotinylated amplification products from all DNA targets and competitors are heat-denatured and hybridized with oligonucleotide probes, which are attached to addressable sets of fluorescent microspheres. The hybrids react with a streptavidin-phycoerythrin conjugate. The microspheres are then analyzed by flow cytometry employing two lasers. A red laser line is used for classification of the microspheres, and a green line excites phycoerythrin, whose fluorescence is related to the concentration of the analyte DNA. As a model, we have developed a multiplex quantitative competitive PCR assay for four targets. The amplification products from targets and competitors (a total of 8 DNA fragments) are determined simultaneously by the multianalyte hybridization assay. The limits of quantification for the hybridization assay of all amplified DNA fragments are below 13 pM. The multiplex quantitative competitive PCR assay detects approximately 500 copies from each target DNA. To our knowledge, the proposed method is the only approach to quantitative PCR that offers such a high potential for multiplexing.     

Waveguide surface plasmon resonance sensor for electrochemically controlled surface reactions

Electrochemical oxidation of the surface of a thin gold film overlaid on an optical waveguide and placed in a cell containing sulfuric acid is monitored by integrated optical surface plasmon resonance (IOSPR) combined with cyclic voltammetry. Waveguide transmittance correlates well with the charge transferred to and from the electrode to oxidize and reduce the surface, with a 60% reduction in transmittance being observed for complete formation of the surface oxide. IOSPR sensors combined with electrochemical measurement and control show potential for sensitive and robust integrated multisensors for electroactive species.     

Chemical labeling for quantitative characterization of surface chemistry

The development and recent achievements of chemical labeling based surface characterization techniques are reviewed. Chemical labeling utilizes the specificity of chemical reactions to attach labeling molecules to surface functionalities, in order to facilitate the detection, identification and quantification of surface species. In this report, different chemical labeling based techniques are compared in terms of sensitivity, specificity and ease of operation. The two most widely used techniques, XPS derivatization and fluorescence labeling, are discussed in detail.     

Bioluminescence DNA hybridization assay for Plasmodium falciparum based on the photoprotein aequorin

A bioluminescence DNA hybridization assay for the detection of Plasmodium falciparum, the most deadly species of malaria, using the photoprotein aequorin as a bioluminescent label has been developed. The current gold standard for the detection of malaria is light microscopy, which can detect down to approximately 50 parasites/microL of blood, but has low-throughput, high costs, and requires high skill, which limit the applicability of the method, especially in the developing regions where malaria detection is mostly needed. The utilization of aequorin as a bioluminescence label offers the advantages of high signal-to-noise ratio and reliable detection down to attomole levels, allowing for the development of highly sensitive and miniaturized high-throughput bioluminescence assays. Herein, we developed a DNA hybridization assay for the detection of P. falciparum based on the competition between the target DNA and the signal generating DNA streptavidin-aequorin for hybridization with the probe DNA. This bioluminescence hybridization assay demonstrated a detection limit of 3 pg/microL and was employed for the detection of target DNA in standard and spiked human serum samples. The DNA hybridization assay was developed in a microplate format without the need for sample PCR amplification, showing the potential suitability of this method in the parallel analysis of samples by low-trained personnel, such as that typically encountered in developing regions.     

A method for quantitative histomorphometric evaluation of soft tissue reactions to implants

The spatial distribution and concentration of distinct cellular elements, and the width of the reaction zone are of particular interest in the evaluation of the compatibility of implant materials. There is a dilemma in the use of interactive histomorphometric evaluation system, At low magnifications cell differentiation is difficult and at high magnification (x400) correct cell differentiation is possible but the evaluated area adjacent to the implant border is small and therefore not necessarily representative. The solution could be the evaluation of random samples at precise localization under high magnification. A light microscope with a software-controlled motorized stage is connected to a personal computer. The software allows definition of one or more polygonal areas at low magnification. A random generator determines the coordinates of the microscopic fields to be analysed and the motorized stage moves automatically to these coordinates. The number of microscopic fields which it is necessary to evaluate in each sample is calculated by the statistical methods described by Stein which take the heterogeneity of the histological structures into account. A software package ranks the various cells at selectable class intervals off the material-tissue interface (distance histogram). Data are stored in ASCII format, which allows importation into any evaluation software. The use of statistical methods seems to be justified for quantitative biocompatibility testing when the tissue encapsulating the implant is heterogeneous and larger than the suitable optical field of the microscope.     

Image analysis system for quantitative metallography

This paper describes a Z-80 microprocessor-based image analyzer developed for global parameter evaluation of images over a 256 × 256 pixel frame. It consists of a microscope,ccd scanner, 6-bit videoadc, Z-80 computer and an image display monitor. Facilities are provided for feature erosion/dilation and halo correction. The paper also presents the details of another more powerful user microprogrammable HP1000 minicomputer-based image analysis system under development. This system consists of an optical microscope/epidiascope coupled to a chalnicon scanner. Here the 512 × 512 pixel image is acquired with 8-bit resolution. It provides for shading correction, auto-delineation, image processing and image analysis functions for evaluation of various basic and derived parameters. Both the systems are software intensive and are realised according to requirements of quantitative metallography. They can also be used for analysis of images obtained in the fields of biology, medicine, geological survey, photography and space.     

On the determination of surface energy from cleavage experiments

An analysis is made of the strain energy distribution in a double-cantilever cleavage specimen of a type which has been used extensively in surface energy determinations. It is shown that the contribution from the uncleaved part of the specimen cannot be neglected as done hitherto by various authors. As a consequence, values for the surface energy of several materials so far determined in cleavage experiments must be revised upward to 50%. The predictions of the theoretical analysis are shown to be confirmed by the results of compliance measurements.

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Zusammenfassung Es wird der Spannungszustand in solchen Probekörpern berechnet, wie sie allgemein für die Bestimmung der Grenzflächenenergie nach der Spaltmethode benutzt werden. Dabei zeigt sich, daß der Beitrag des ungespaltenen Probeteils zur insgesamt gespeicherten elastischen Energie keineswegs vernachlässigt werden darf, wie bisher von zahlreichen Autoren angenommen wurde. Dies hat zur Folge, daß alle bisher nach der Spaltmethode bestimmten Werte der spezifischen Grenzflächenenergie um his zu 50% nach oben korrigiert werden müssen. Die theoretische Aussage konnte durch sogenannte Compliance-Messungen bestätigt werden.

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Résumé On a calculé l'état de contrainte d'une éprouvette du type de celles utilisées pour les déterminations d'énergies de surface par la méthode de la fente. Il apparait dans ces conditions que la contribution de la partie non fondue de l'éprou-vette dans l'estimation de l'energie élastique emmagasinée totale n'est en aucun cas négligeable, comme de nombreux auteurs font jusqu'à présent admis. Par suite, toutes les valeurs trouvées jusqu'à present pour les energies de surface sont trop faibles et doivent être augmentées dans une proportion atteignant 50%. La prevision théorique put être vérifiée par Compliance measurements.

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List of Symbols A cross-sectional area - A _i free constants, see (15) - B _i free constants, see (15) - C compliance - E modulus of elasticity - F external force - G shear modulus - J moment of inertia of the cross section - L crack length - L ₁ length of the uncleaved part of the specimen - m Poisson's ratio - N ₁, N ₂ shortened terms, see (16) - r, polar coordinates - r _a external radius of the semiannular disc - r _i internal radius - S surface energy - t cross-sectional height - U stored elastic energy - w cross-sectional width - W externally supplied energy - x correction factor - y shortened term, see (25) - y specific surface energy - ₀ displacement at the point of application of the force - shortened term, see (20) - _r, _t normal stresses - _r shear stresses - stress function     

The kinetics of surface monomolecular reactions

A comparison of various theoretical kinetic schemes with experimental results shows that the surface monomolecular decay of both individual molecules and the fragments of polymolecular complexes is a nonequilibrium process corresponding to first-and second-order reactions, respectively.     

Decoupling co-existing surface plasmon polariton (SPP) modes in a nanowire plasmonic waveguide for quantitative mode analysis

Knowledge of surface plasmon polariton (SPP) modes in one-dimensional (1D) metallic nanostructures is essential for the development of subwavelength optical devices such as photonic circuits,integrated light sources,and photodetectors.Despite many efforts to characterize the propagation parameters of these subwavelength 1D plasmonic waveguides,such as Ag nanowires,large discrepancies exist among available reports owing to their sensitivity to the relative weights of co-existing SPP modes and the lack of a method of decoupling these modes and analyzing them separately.In this work,we develop an interference method to distinguish different SPP modes that are simultaneously excited in a Ag nanowire waveguide and measure their propagation parameters separately.By extracting information from the propagation-distancedependent intensity oscillations of the scattered light from the nanowire tip,the effective refractive indices,propagation lengths,and relative mode weights of co-existing SPP modes supported by the nanowire are derived from a mode interference model.These parameters depend strongly on the nanowire diameter and excitation wavelength.In particular,we demonstrate the possibility of selective excitation of different SPP modes by varying the nanowire diameter.This new mode analysis technique provides unique insights into the development and optimization of SPP-based applications.     

Ultrafast kinetic DNA hybridization assay based on the visualization of threshold turbidity

We report herein the development of an ultrafast kinetic DNA hybridization assay system based on the visualization of threshold turbidity associated with the assembly of polystyrene nanospheres. Initial testing of our diagnostic protocol on a sequence associated with the anthrax lethal factor indicates that a visually identifiable, turbidity-definitive, and kinetic threshold state could be reached at a time as short as 1 min. The assay scheme allows for both target concentration quantification and differentiation of single base mismatches through registry of the threshold turbidity onset time. The positively charged environment on nanospheres not only contributes to expedited signal generation but also imparts cooperative DNA binding properties. The kinetic visual protocol complements conventionally used thermodynamic strategies and provides an entry point for the circumvention of assay issues associated with ill-defined thermodynamic end points.     

Electrochemical cell for surface analysis

We present a simple cost-effective design for an HOPG electrode that is well suited to voltammetric experiments accompanied by surface analysis. The utility of the electrode is demonstrated by an AFM study on the morphology of electrodeposited MoO2 nanowires. The design can be applied to a wide range of electrode materials.     

Poly(dimethylsiloxane) surface modification by plasma treatment for DNA hybridization applications

In this work, the surface modification of poly(dimethylsiloxane) (PDMS) was carried out by using a 2-step plasma modification with Ar followed by acrylic acid (AAc). The optimal conditions were found to be 0.5 min with Ar at 0.7 mbar; and 5 min with AAc at 0.2 mbar. The water contact angle (WCA) of the native PDMS decreased from 110 degrees to 30 degrees after modification, then stabilized to values between 50 degrees to 60 degrees after 1 day exposure to air. The stability of the modified PDMS was further improved by Soxhlet-extracting the PDMS with hexane prior to plasma treatment. Atomic force microscopy (AFM) showed significant changes in surface morphology after the 2-step plasma modification. X-ray photoelectron (XPS) spectroscopy further confirmed the successful modification of the PDMS surface with PAAc, by exhibiting C1s peaks at 285.9 eV, 287.4 eV and 289.9 eV, originating from C-O, C=O and O-C=O moieties, respectively. Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy of the poly(acrylic acid) (PAAc) modified PDMS surface showed a distinctive peak at 1715 cm(-1), attributed to the presence of COOH groups from the PAAc. The carboxyl peak on the spectra of the PAAc modified PDMS was quite stable even after storage at room temperature in phosphate buffer saline (PBS) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer for 17 h. 5'-amino-terminated oligonucleotides were covalently attached to the PAAc modified PDMS surface via carbodiimide coupling. Subsequently, fluorescently tagged complementary oligonucleotides were successfully hybridized to this surface, as determined by fluorescence microscopy.