An integrated biosensor platform for extraction and detection of nucleic acids

The development of portable systems for analysis of nucleic acids (NAs) is crucial for the evolution of biosensing in the context of future healthcare technologies. The integration of NA extraction, purification, and detection modules, properly actuated by microfluidics technologies, is a key point for the development of portable diagnostic systems. In this paper, we describe an integrated biosensor platform based on a silicon–plastic hybrid lab-on-disk technology capable of managing NA extraction, purification, and detection processes in an integrated format. The sample preparation process is performed by solid-phase extraction technology using magnetic beads on a plastic disk, while detection is done through quantitative real-time polymerase chain reaction (qRT-PCR) on a miniaturized silicon device. The movement of sample and reagents is actuated by a centrifugal force induced by a disk actuator instrument. The assessment of the NA extraction and detection performance has been carried out by using hepatitis B virus (HBV) DNA genome as a biological target. The quantification of the qRT-PCR chip in the hybrid disk showed an improvement in sensitivity with respect to the qRT-PCR commercial platforms, which means an optimization of time and cost. Limit of detection and limit of quantification values of about 8 cps/reaction and 26 cps/reaction, respectively, were found by using analytical samples (synthetic clone), while the results with real samples (serum with spiked HBV genome) indicate that the system performs as well as the standard methods.     

Piezoelectric biosensors: strategies for coupling nucleic acids to piezoelectric devices

The development of a piezoelectric biosensor based on nucleic acids interaction is presented focusing on the methodology for probe immobilization. This is a key step in any DNA biosensor development. Often, the detection limits and, in general, the analytical performances of the biosensor can be improved by optimizing the immobilization of the receptor on the transducer surface. DNA must be attached to the solid support, retaining native conformation, and binding activity. This attachment must be stable over the course of a binding assay and, in addition, sufficient binding sites must be presented to the solution phase to interact with the analyte. In this paper, the optimization of the coating of the gold quartz crystal surface, to immobilize an oligonucleotide probe, is reported. Two immobilization procedures are illustrated in details with a comparison regarding the immobilization of the probe, the detection of the hybridization reaction, and the possibility of regeneration. The two procedures are based on the use of biotinylated or thiolated DNA probes. Specific applications will be also presented.     

Single-molecule,hybridization-based strategies for short nucleic acids detection and recognition with nanopores

DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore- and hybridization-based DNA detection, and explores a range of state-of-the-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.     

Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction

Here we have developed a sensitive DNA amplified detection method based on isothermal strand-displacement polymerization reaction. This method takes advantage of both the hybridization property of DNA and the strand-displacement property of polymerase. Importantly, we demonstrate that our method produces a circular polymerization reaction activated by the target, which essentially allows it to self-detect. Functionally, this DNA system consists of a hairpin fluorescence probe, a short primer and polymerase. Upon recognition and hybridization with the target ssDNA, the stem of the hairpin probe is opened, after which the opened probe anneals with the primer and triggers the polymerization reaction. During this process of the polymerization reaction, a complementary DNA is synthesized and the hybridized target is displaced. Finally, the displaced target recognizes and hybridizes with another probe, triggering the next round of polymerization reaction, reaching a target detection limit of 6.4 × 10⁻¹⁵ M.     

Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.

A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.     

Validation of astrobiology technologies and instrument operations in terrestrial analogue environments

Terrestrial analogue environments are places on Earth that present geological or environmental conditions that are similar to those found on an extraterrestrial body. Analogue environments serve four functions: (1) learn about planetary processes on Earth and elsewhere; (2) test technologies, methodologies, and protocols; (3) train highly-qualified personnel, as well as science and operations teams; (4) engage the public, space agencies, media, and educators. Analogue studies also enable the development and validation of biosignatures and detection techniques. Analogue programs include the Canadian Space Agency's Canadian Analogue Research Network, NASA's Astrobiology Science and Technology for Exploring Planets, and NASA's Analog Missions. Examples of technology and instrument testing and validation in analogue environments include the Haughton-Mars Project Research Station, the Arctic Mars Analog Svalbard Expedition (AMASE), the Rio Tinto basin, and NASA's Field Integrated Design and Operations (FIDO).     

Origin of life: Consideration of alternatives to proteins and nucleic acids

Summary Starting with relatively simple, non-hydrolyzable compounds in aqueous solution, entirely spontaneous condensations give rise to polymers that contain purines, pyrimidines, amino acids, coenzymes, lipid components and even phosphate. The presence of certain lipid micelles allows significant product formation at millimolar substrate concentrations. The first step involves formation of a Michael adduct from--unsaturated carbonyl compounds and various nucleophiles. Polymerization of these adducts occurs via sequential Knoevenagel condensations. All reactions take place readily at temperatures below 45°. The polymers can act as macromolecular catalysts as evidenced by hydrolytic activity. The purines and pyrimidines in the polymers appear to be capable of both base pairing and stacking interactions with ribonucleic acids. Specific examples of potential alternatives to base pairing are presented. These results are discussed from the standpoint of the spontaneous development of reproducing molecules. Proteins and nucleic acids may be evolutionary developments which have displaced earlier biopolymers.     

Metagenomics of microbial and viral life in terrestrial geothermal environments

Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme- and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes.     

New approaches to in situ detection of nucleic acids

The present paper reviews recent results obtained by different molecular biology-based, immunocytological approaches to the localization and identification of nucleic acids in sections of biological material. Examples of sensitive, high-resolution detection methods for RNA, DNA or specialized DNA regions are presented. Special emphasis is placed on the potential values and limitations of these new methods.Presented at the XXXVII Symposium of the Society for Histochemistry, 23 September 1995, Rigi Kaltbad, Switzerland     

An instrument design for non-contact detection of biomolecules and minerals on Mars using fluorescence

We discuss fluorescence as a method to detect polycyclic aromatic hydrocarbons and other organic molecules, as well as minerals on the surface of Mars. We present an instrument design that is adapted from the ChemCam instrument which is currently on the Mars Science Lander Rover Curiosity and thus most of the primary components are currently flight qualified for Mars surface operations, significantly reducing development costs. The major change compared to ChemCam is the frequency multipliers of the 1064 nm laser to wavelengths suitable for fluorescence excitation (266 nm, 355 nm, and 532 nm). We present fluorescence spectrum for a variety of organics and minerals relevant to the surface of Mars. Preliminary results show minerals already known on Mars, such as perchlorate, fluoresce strongest when excited by 355 nm. Also we demonstrate that polycyclic aromatic hydrocarbons, such as those present in Martian meteorites, are highly fluorescent at wavelengths in the ultraviolet (266 nm, 355 nm), but not as much in the visible (532 nm). We conclude that fluorescence can be an important method for Mars applications and standoff detection of organics and minerals. The instrument approach described in this paper builds on existing hardware and offers high scientific return for minimal cost for future missions.     

Electric chips for rapid detection and quantification of nucleic acids

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.     

Automated system for capture and detection of nucleic acids.

A fully automated nucleic acid analysis system is described, which offers positive sample identification, improved sensitivity and reduced user interaction compared to conventional techniques. The system relies on the sequence-specific capture of DNA onto solid-phase particles, confirming product identity without the problems of interpretation and lack of sequence information inherent in gel-based analyses. The system can be used for sequence confirmation, mutation analysis and semiquantitative detection of PCR products.     

New primers for the class Actinobacteria: application to marine and terrestrial environments

In this study, we redesigned and evaluated primers for the class Actinobacteria. In silico testing showed that the primers had a perfect match with 82% of genera in the class Actinobacteria, representing a 26-213% improvement over previously reported primers. Only 4% of genera that displayed mismatches did so in the terminal three bases of the 3' end, which is most critical for polymerase chain reaction success. The primers, designated S-C-Act-0235-a-S-20 and S-C-Act-0878-a-A-19, amplified an approximately 640 bp stretch of the 16S rRNA gene from all actinobacteria tested (except Rubrobacter radiotolerans) up to an annealing temperature of 72 degrees C. An Actinobacteria Amplification Resource (http://microbe2.ncl.ac.uk/MMB/AAR.htm) was generated to provide a visual guide to aid the amplification of actinobacterial 16S rDNA. Application of the primers to DNA extracted from marine and terrestrial samples revealed the presence of actinobacteria that have not been described previously. The use of 16S rDNA similarity and DNA-DNA pairing correlations showed that almost every actinomycete clone represented either a new species or a novel genus. The results of this study reinforce the proposition that current culture-based techniques drastically underestimate the diversity of Actinobacteria in the environment and highlight the need to evaluate taxon-specific primers regularly in line with improvements in databases holding 16S rDNA sequences.     

Probe design rules and effective enzymes for endonuclease-based detection of nucleic acids

Junction probe (JP) platform is an isothermal endonuclease-based detection assay for both RNA and DNA. Herein, we screen 31 REAse and identify effective restriction endonucleases that can be used for JP detection. Secondly, we investigate how different probe architectures affect JP cleavage rates and conclude that although molecular beacon (MB) JP probes give less background noise than linear JP probes, the cleavage of MB JP probes are slower than linear JP probes.     

Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.     

Use of acid,alkali and enzymes for the extraction of nucleic acids from pollen

Various methods for extracting nucleic acids from pollen were tested to find a suitable procedure for obtaining a pure preparation of nucleic acids uncontaminated by polysaccharides and polyphosphates without the use of ion exchangers. Extraction was carried out with perchloric acid, potassium hydroxide, ribonuclease and deoxyribonuclease, sodium tetraborate, and combinations of these. In all fractions, residues of precipitates and residues of extracted pollen matter, the quantity of RNA, DNA, proteins and concomitants,i.e. Polysaccharides and polyphosphates, was determined. The purity of preparations was checked by means of UV-spectra. The criterion of nucleic acid purity was agreement between the nucleic acid amounts calculated on the basis of measurement of absorption in UV-region, orcinol reaction and content of phosphorus. It was found that in our material only a few methods would be applicable and with great limitation, because many polysaccharides and polyphosphates appeared in nucleic acid fractions.     

Molecular colony diagnostics: detection and quantitation of viral nucleic acids by in-gel PCR

When PCR is carried out in a polyacrylamide gel, each target molecule forms a molecular colony that comprises many copies of the original template. By counting the number of colonies, one can directly determine the target titer, with 100% of the DNA molecules and approximately 15% of the RNA molecules being detected. Furthermore, because of the spatial separation of the products in the gel, no interference is observedfrom another simultaneously amplified target even if it is present at a 106 higher amount orfrom human nucleic acids that outweigh the target by up to a factor of 1,012, which is often true of clinical samples. All these features provide for an accurate and reliable assay of viruses even at very low amounts, that is, in cases most important to diagnostics.     

Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil

A universally adaptable protocol for quantitative extraction of high-purity nucleic acids from soil is presented. A major problem regarding the extraction of nucleic acids from soil is the presence of humic substances, which interfere with the extraction process itself and in subsequent analytical manipulations. By the approach described here, the humic compounds are precipitated prior to cell lysis with Al(2)(SO(4))(3), and thus eliminated prior to the nucleic acid extraction. The protocol allows for removing of a considerable content and range of humic acids and should therefore be applicable for a wide spectrum of soil types. Accordingly, reproducible results in analyses of different soil types are made possible, inclusively for quantitative comparisons.     

A simple and sensitive method for detection of nucleic acids fixed on nylon-based filters

Two methods for detection of nucleic acids, fixed on nylon membranes, are described. A negative staining, using the Auro-dye technique described for proteins, results in a sensitive and fast detection of nucleic acids fixed on nylon membranes, which are routinely used in molecular biology. Subsequent hybridization treatment does not influence the staining. A positive staining with a cationic iron colloid is also described. Sensitivity and application of both methods are discussed.