Sensitive quantitative nucleic acid detection using oligonucleotide microarrays

We report a new theoretical approach to optimize the performance and quantify the results of gene expression oligonucleotide microarrays which are widely used in biomedical research. An on-array hybridization isotherm that takes into account the screened Coulomb repulsion between the assayed nucleic acid target and the layer of surface tethered oligonucleotide probes is presented. The hybridization efficiency is found as a function of the genomic target (sequence, length, and concentration), array parameters (probe sequence and length, surface probe density), and hybridization conditions (temperature and buffer ionic strength). We present simple relations for the hybridization signal maximum and the linear dynamic detection range and show explicit criteria for optimization. The approach is based on an extension of our recently published theory (Vainrub, A.; Pettitt, B. M. Phys. Rev. E 2002, 66, art. no.-041905) which we generalize here for the cases of target depletion effects and arbitrary target length.     

Molecular interactions of plant and algal polysaccharides

This article presents an overview of the interaction in solution of certain plant and algal polysaccharides with cations and also of their interaction in admixture with other polysaccharides and proteins. The mechanism of gelation of pectin and alginate in the presence of calcium ions is discussed together with the specific binding of potassium ions to kappa carrageenan and its influence on the coil–helix transition. The associative and segregative phase behaviour that is encountered in aqueous solutions of mixed polysaccharide systems is considered together with the formation of the soluble and insoluble complexes in mixtures of polysaccharides and proteins.     

Enzyme inhibition-based biosensor for the electrochemical detection of microcystins in natural blooms of cyanobacteria

An electrochemical biosensor for the detection of microcystin has been developed based on the inhibition of the protein phosphatase 2A (PP2A) by this cyanobacterial toxin. The enzyme has been immobilised by entrapment using a poly(vinyl alcohol) azide-unit pendant water-soluble photopolymer (PVA-AWP). Electrode supports and immobilisation conditions have been optimised by colorimetric assays, the highest immobilisation yields being obtained with screen-printed graphite electrodes and the 1:2 PP2A:PVA ratio. Catechyl monophosphate (CMP), α-naphthyl phosphate (α-NP) and 4-methylumbelliferyl phosphate (4-MUP) have been used as phosphorylated substrates to monitor the protein phosphatase activity by electrochemical methods, the former providing the highest chronoamperometric currents at appropriate working potentials (+450 mV versus Ag/AgCl). Incubation with standard microcystin solutions has demonstrated the inhibition of the immobilised enzyme, proportional to the toxin concentration. The standard inhibition curve has provided a 50% inhibition coefficient (IC₅₀) of 83 μg L⁻¹, a limit of detection (LOD; 35% inhibition) of 37 μg L⁻¹, and 100% inhibition at about 1000 μg L⁻¹. Real samples of cyanobacterial blooms from the Tarn River (Midi-Pyrénées, France) have been analysed using the developed amperometric biosensor and the toxin contents have been compared to those obtained by a conventional colorimetric protein phosphatase inhibition (PPI) assay and high-performance liquid chromatography (HPLC). The results clearly justify the use of the developed amperometric biosensor as screening method for microcystin detection.     

Multiplexed analyte and oligonucleotide detection on microarrays using several redox enzymes in conjunction with electrochemical detection

We show that multiple enzyme tags may be used in an immunoassay format or for the detection of sequence-specific DNA on microarrays. The assays may be multiplexed and monitored under separate solution and voltage differences. Thus, the detection method relies on an electrochemical detection format, whereby multiple enzymes can be sensed. In our case we utilize horseradish peroxidase, laccase, and glucose dehydrogenase as enzymes attached to specific antibodies or to streptavidin.     

A new approach to oligonucleotide labelling using Diels-Alder cycloadditions and detection by SERRS

Diels-Alder cycloaddition has been used to add a benzotriazole azo dye to a diene tagged oligonucleotide to generate unique SERRS signals at 10 attomoles.     

A simple and rapid detection of viral protein using RNA oligonucleotide in a biosensor

The monitoring of non-structural viral protein 3 (NS3) has been of considerable interest in developing simple and reliable methods for detection of hepatitis C virus (HCV) for applications in diagnostic medicine. Although enzyme-linked immunosorbent assay (ELISA) is the most general method in HCV detection, using antibody brings problems. This method is temperature-sensitive and requires specific reactions condition. In addition, secondary antibody conjugated with enzyme and fluorescent dye is required. To overcome these bottlenecks, we designed a streptavidin-biotin conjugation method, namely, the RNA oligonucleotide sensor system that could monitor viral protein with detection limit of 500 pg/mL by using biotin-tagged RNA oligonucleotide in forteBio??s Octet optical biosensor system. In this study, we proposed an efficient method for simple and convenient detection of HCV viral protein, with the advantage of target specific monitoring.     

Review and assessment of in vitro detection methods for algal toxins

Algal toxins produced by marine and freshwater microalgae present a significant analytical challenge because of their complex structures and frequent occurrence as mixtures of structural congeners, which differ in toxic potencies and are present at varying proportions in contaminated samples. Rapid, sensitive in vitro detection methods specific for each class of algal toxins have been developed over the past decade, including immunoassays, enzyme inhibition assays, receptor assays, and cell assays. This review discusses the conceptual approaches to assay development and provides a detailed assessment of the use of in vitro detection methods for marine and freshwater algal toxins.     

New flavin and deazaflavin oligonucleotide conjugates for the amperometric detection of DNA hybridization

The functionalization of an oligonucleotide by flavin and deazaflavin derivatives allowed an amperometric determination of the hybridization process through the disappearance of the electroactivity of the free oligonucleotide and the appearance of a new electrochemical signal characteristic of the resulting duplex.     

Rapid detection of algal toxins by microfluidic immunoassay

Herein we report fabricating a microfluidic device to monitor harmful algal blooming (HAB). The heterogeneous immuno-enzyme assay was integrated into a self-designed microfluidic chip for rapid and automatic analysis of algal toxins. The device was made from polydimethylsiloxane (PDMS) and was assembled with a home-made control system. The performance of the system was demonstrated by the detection of microcystin, saxitoxin and cylindrospermopsin, the major cyanotoxins. In one single microfluidic chip, multiple samples were controlled and analysed in a parallel manner. Under the optimal conditions, the linear range and the limit of detection of microcystins were 0-5.0 ng mL(-1) and 0.02 ng mL(-1) respectively. The total analysis time was less than 25 min. The designed device was highly automatic, more efficient and economic compared to conventional techniques.     

Real-time observation of temperature-dependent protein-protein interactions using real-time dual-color detection system

This study examined the ability of a real-time dual-color detection system to allow direct observations of the kinetics of temperature-dependent protein-protein interaction at a single-molecule level. The primary target protein was an Alexa Fluor^® 488-labeled actin conjugate, which had been pre-incubated with an unlabeled rabbit anti-actin antibody (IgG). The complementary fluorescent protein was Alexa Fluor^® 633-labeled goat anti-rabbit IgG antibody, which interacts with the rabbit anti-actin antibody (IgG) bound to the Alexa Fluor^® 488-labeled actin conjugate. The individual protein molecules labeled with different fluorescent dyes in solution were effectively focused, interacted with the other protein molecules at 500 aM, and detected directly in real-time using the dual-wavelength (λ_ex = 488 and 635 nm) laser-induced fluorescence detection system. The kinetics of the protein-protein interactions were examined at different temperatures (12-32 °C). At concentrations in the aM range, the number of bound complex molecules through the protein-protein interaction decreased gradually with time at a given temperature, and increased with decreasing temperature at a set time. A high concentration (above 500 pM) of the protein sample caused aggregation and nonspecific binding of the protein molecules, even though the protein molecules were not an example of complementary binding. The results demonstrated that the real-time kinetics of a protein-protein interaction could be analyzed effectively at the single-molecule level without any time delay using the real-time dual-color detection system.     

Label-free detection of neuron-drug interactions using acoustic and Kelvin vibrational fields

Kelvin and acoustic fields of high-frequency have been employed in the non-invasive investigation of immortalized hypothalamic neurons, in order to assess their response to different concentrations of specific drugs, toxins, a stress-reducing hormone and neurotrophic factors. In an analytical systems biology approach, this work constitutes a first study of living neuron cultures by scanning Kelvin nanoprobe (SKN) and thickness shear mode (TSM) acoustic wave techniques. N-38 hypothalamic mouse neurons were immobilized on the gold electrode of 9 MHz TSM acoustic wave devices and gold-coated slides for study by SKN. The neurons were exposed to the neurochemicals betaseron, forskolin, TCAP, and cerebrolysin. Signals were collected with the TSM in real-time mode, and with the SKN in scanning and real-time modes, as the drugs were applied at biologically significant concentrations. With the TSM, for all drugs, some frequency and resistance shifts were in the same direction, contrary to normal functioning for this type of instrument. Possible mechanisms are presented to explain this behaviour. An oscillatory signal with periodicity of approximately 2 min was observed for some neuron-coated surfaces, where the amplitude of these oscillations was altered upon application of certain neurotrophic factors. These two new techniques present novel and non-invasive electrodeless methods for detecting changes at the cellular level caused by a variety of neuroactive compounds, without killing or destroying the neurons.     

Innovative detection methods for aquatic algal toxins and their presence in the food chain

Detection of aquatic algal toxins has become critical for the protection of human health. During the last 5 years, techniques such as optical, electrochemical, and piezoelectric biosensors or fluorescent-microsphere-based assays have been developed for the detection of aquatic algal toxins, in addition to optimization of existing techniques, to achieve higher sensitivities, specificity, and speed or multidetection. New toxins have also been incorporated in the array of analytical and biological methods. The impact of the former innovation on this field is highlighted by recent changes in legal regulations, with liquid chromatography–mass spectrometry becoming the official reference method for marine lipophilic toxins and replacing the mouse bioassay in many countries. This review summarizes the large international effort to provide routine testing laboratories with fast, sensitive, high-throughput, multitoxin, validated methods for the screening of seafood, algae, and water samples.     

Analysis of the factors affecting the accuracy of detection for single base alterations by oligonucleotide microarray

The oligonucleotide microarray, a high-throughput polymorphism detection technology, holds great promise for the characterization of complex genetic variance. To achieve greater sensitivity and specificity for it to be an effective platform technology we present results and discuss some of the factors influencing signal intensities and single-mismatch discrimination in array-based mutation/SNP detection. Probes with a series of concentrations were spotted onto the slide in order to find the optimal concentration with the identifiable satisfying signals and the stable ratios between matched and mismatched probes. It was found that under our experimental conditions, when the initial probe concentration is higher than the maximum immobilization capability of the slide (7.5 microM), the hybridization signal will be saturated and the ratio between matched and mismatched probes will be more stable than at a lower probe concentration. Considering the cost of probes and the systematic stability, a constant spotting concentration of 10 microM was selected. The stability of different types of mismatched oligo-DNA duplexes on the glass surface was also confirmed. The results show that the order of stability of mismatched oligo-DNA duplexes on a glass surface is in general agreement with previous reports conducted using liquid and polyacrylamide gel pads. This suggests that the influence of the mismatched base pair on the stability of the duplex in a solid hybridization system is similar to that in the solution hybridization environment.     

Surfactant bilayers for the direct electrochemical detection of affinity interactions

Simple methods of preparing the direct affinity sensors are proposed. Due to the self-consistent introduction of a hydrocarbon chain bound with oligonucleotide pentadecathymidylate (dT(15)) into the hydrophobic region of surfactant bilayer or the adsorption of antibodies on the bilayer surface, the immobilizations of oligonucleotide or antibodies were carried out correspondingly. The responses were detected by impedance spectroscopy. Whereas the specific DNA-coupling caused the decrease of real part of impedance, the antibody-antigen interaction caused the increase of real part. The obtained results give an opportunity for the development of impedimetric affinity sensors for clinical analysis or for the detection of various environmental pollutants.     

Towards multi-colour strategies for the detection of oligonucleotide hybridization using quantum dots as energy donors in fluorescence resonance energy transfer (FRET)

The potential for a simultaneous two-colour diagnostic scheme for nucleic acids operating on the basis of fluorescence resonance energy transfer (FRET) has been demonstrated. Upon ultraviolet excitation, two-colours of CdSe/ZnS quantum dots with conjugated oligonucleotide probes act as energy donors yielding FRET-sensitized acceptor emission upon hybridization with fluorophore (Cy3 and Alexa647) labeled target oligonucleotides. Energy transfer efficiencies, Förster distances, changes in quantum yield and lifetime, and signal-to-noise with respect to non-specific adsorption have been investigated. The dynamic range and limit-of-detection are tunable with the concentration of QD-DNA conjugate. The Cy3 and Alexa647 acceptor schemes can detect target from 4 to 100% or 10 to 100% of the QD-DNA conjugate concentration, respectively. Nanomolar limits of detection have been demonstrated in this paper, however, results indicate that picomolar detection limits can be achieved with standard instrumentation. The use of an intercalating dye (ethidium bromide) as an acceptor to alleviate non-specific adsorption is also described and increases signal-to-noise from S/N < 2 to S/N = 9-10. The ethidium bromide system had a dynamic range from 8 to 100% of the QD-DNA conjugate concentration and could detect target in a matrix containing an excess of non-complementary nucleic acid.     

Approaches to the liquid chromatographic determination of carboxylic acids using potentiometric detection with a metallic copper electrode

Summary A metallic copper electrode is evaluated as a potentiometric detector for carboxylic acids. The application of this device to ion-exchange chromatography is illustrated by the determination of oxalate in urine. Oxalate was selectively detected in the presence of a 100-fold excess of sulphate after separation on a low-capacity methacrylate anion-exchange column using 0.7mM potassium hydrogen phthalate at pH 7.1 as eluent. Calibration plots were linear up to 50ppm of oxalate. Potentiometric detection has also been applied to ion-exclusion chromatography using 0.005% phosphoric acid as eluent. With this method detection limits of 0.2, 2.1, 5.0 and 5.3μg were obtained for formic, acetic, propionic and iso-butyric acids, respectively.     

Mapping the placement of oligonucleotide molecules using scanning probe microscopy

The successful development of novel bio-inspired devices requires the ability to place specific biomolecules on a substrate with nanometre precision, in such a way so that their bioactivity is retained. A method is required that can verify this bio-modification. Scanning probe microscopy (SPM) can image and probe a surface in a liquid environment with nanometre resolution. Using short chain complementary oligonucleotides as the bioactive molecules we have modified continuous and patterned gold substrates and SPM probes. We demonstrated that the attached oligonucleotides retained their biological activity after surface attachment with a hybridization interaction force that varies between 50 and 400pN as measured by SPM force measurements. Finally, the position of the attached oligonucleotides was determined with nanometre resolution. Thus we have demonstrated the capabilities of SPM in the application of the development of substrates and templates suitable for forming the basis of novel and innovative devices.     

Non-radioactive oligonucleotide probe for detection of clinical enterotoxigenic Escherichia Coli isolates of bovine origin

Oligonucleotides (32 or 34 mer) corresponding to enterotoxigenic Escherichia coli ST_Ia (ST-P) toxin were tailed with Bio-11-dUTP using terminal deoxynucleotidyl transferase. Plasmids from clinica E.Coli isolates were prepared by modified rapid alkaline lysis procedure and dot-spotted. Biotinylated oligonucleotide probes were hybridized to detect the St_Ia toxin gene. The results were in agreement with that obtained by radioactive oligonucleotide probes. Of 135 clinical isolates (sampled from 6 different regions of France), only 7 (5.2 %) were found to be ST_Ia⁺. These 7 isolates were the only ones to be found positive for the K99 adhesive pili antigen. Both the proves were specific to the ST_Ia toxin gene and failed to detect the closely related ST_Ib (ST-H) toxin gene. Possibilities of their wide usage in clinical labs are discussed.     

Quantitation of ultraviolet-induced single-strand breaks using oligonucleotide chip

A simple, accurate and robust methodology was established for the direct quantification of ultraviolet (UV)-induced single-strand break (SSB) using oligonucleotide chip. Oligonucleotide chips were fabricated by covalently anchoring the fluorescent-labeled ssDNAs onto silicon dioxide chip surfaces. Assuming that the possibility of more than one UV-induced SSB to be generated in a small oligonucleotide is extremely low, SSB formation was investigated quantifying the endpoint probe density by fluorescence measurement upon UV irradiation. The SSB yields obtained based on the highly sensitive laser-induced fluorometric determination of fluorophore-labeled oligonucleotides were found to coincide well with that predicted from a theoretical extrapolation of the results obtained for plasmid DNAs using conventional agarose gel electrophoresis. The developed method has the potential to serve as a high throughput, sample-thrifty, and time saving tool to realize more realistic, and direct quantification of radiation and chemical-induced strand breaks. It will be especially useful for determining the frequency of SSBs or lesions convertible to SSBs by specific cleaving reagents or enzymes.     

Hybridization of oligonucleotide by using DNA self-assembled monolayer

The interaction between DNA immobilized on surface and oligonucleotides at the interface is important in detection and diagnostic processes. However, it is difficult to immobilize DNA with maintaining its activity and to realize an efficient hybridization in previous methods. Here, to establish a novel DNA-functionalized surface, the DNA self-assembled monolayer (SAM) was constructed on a gold substrate using thiolated DNA composed of double-stranded (ds) and single-stranded (ss) portion. The DNA SAM was characterized by surface plasmon resonance (SPR), XPS. The hybridization of ss portion of DNA was attempted using the SAM, and in situ monitored by SPR. XPS measurement indicated that the thiolated DNA could form a stable monolayer on a gold substrate through sulfur–gold interaction. SPR measurement implied that the long axis of the DNA standing on the substrate. These results indicated formation of the DNA SAM on the substrate. Hybridization of target DNA containing a complementary sequence for the probe portion was observed by SPR. Moreover, one mismatch of oligonucleotide could be distinguished using the DNA SAM. The SPR result indicates that hybridization of target DNA and probe DNA on the DNA SAM occurs on the DNA SAM.