Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection

A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng microl(-1) and 0.45 ng microl(-1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.     

Detection of SEB gene by bilayer lipid membranes nucleic acid biosensor supported by modified patch-clamp pipette electrode

This work reports a kind of novel bilayer lipid membranes (BLMs) nucleic acid biosensor supported by modified patch-clamp pipette electrode was developed to detect staphylococcus enterotoxins B (SEB) gene. BLMs were formed within 15 min and able to be operated at least 24 h. Hydrophobic dodecane tail (C12) modified 18 bp single-stranded DNA (ssDNA) probe was immobilized on BLMs. The electrochemical currents versus the different concentration of ssDNA probe immobilized on BLMs indicated linearly correlation. The BLMs nucleic acid biosensor was fabricated by selecting the ssDNA probe as the signal sensing element with the concentration of 273.65 ng/mL. The electrochemical performance of the biosensor for the detection of SEB was investigated. The result showed that linear relationship was found between the current and ln(concentration) from 20 to 5000 ng/mL and the detection limit was 20 ng/mL. In addition, the biosensor was specific response to SEB gene and showed no significant current alteration in electrolyte which containing no SEB gene. Finally, Atom Force Microscope (AFM) images could be observed and used to evaluate the superficial microstructure of BLMs, ssDNA immobilized on BLMs and BLMs after hybridization. The BLMs nucleic acid biosensor supported by modified patch-clamp pipette electrode will become a highly sensitive, rapid, selective analytical tool for detection of Staphylococcus aureus, which produce SEB.     

Synthesis of a novel fluorescent probe useful for DNA detection

In this paper, a novel fluorescent probe 2-methylbenzo[b][1,10] phenanthrolin-7(12H)-one (m-BPO) is synthesized, and its molecular structure has been characterized by IR, UV, MS, (1)H-NMR and elements analysis. The fluorescent characteristics of m-BPO were investigated in detail. It was found that DNA had the ability to quench the fluorescence of m-BPO at 411 nm (lambda(ex)=286 nm), and the quenched intensity of fluorescence was proportional to the concentration of DNA. Based on this fact, m-BPO has been used as the fluorescent probe for detection of calf thymus DNA (ctDNA) and fish semen DNA (fsDNA). Under the optimal conditions, the calibration curves are linear up to 15.0 microg/ml for both ctDNA and fsDNA. The corresponding detection limits are 3.6 ng/ml for ctDNA and 5.5 ng/ml for fsDNA, respectively. The interaction mechanism for the binding of m-BPO to ctDNA was studied in detail, and the results suggested that the interaction mode between m-BPO and ctDNA was groove binding.     

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.     

A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

Whole-cell biosensors have become popular tools for detection of ecotoxic compounds in environmental samples. We have developed an assay optimized for flow cytometry with detection of genotoxic compounds in mind. The assay features extended pre-incubation and a cell density of only 10(6)-10(7) cells/mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS-induction in whole soil samples. Soil microcosms were spiked with a dilution-series of crude broth extract from the mitomycin C-producing streptomycete Streptomyces caespitosus. Biosensors extracted from these microcosms after 1 day of incubation at 30 degrees C were easily distinguished from extracts of non-contaminated soil particles when using flow cytometry, and induction of the biosensor by mitomycin C was detectable at concentrations as low as 2.5 ng/g of soil.     

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 μg/L, with 100 μg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.     

Electrochemical measurement of DNA hybridization using nanosilver as label and horseradish peroxidase as enhancer

A simple and sensitive electrochemical DNA biosensor based on in situ DNA amplification with nanosilver as label and horseradish peroxide (HRP) as enhancer has been designed. The thiolated oligomer single-stranded DNA (ssDNA) was initially directly immobilized on a gold electrode, and quartz crystal microbalance (QCM) gave the specific amount of ssDNA adsorption of 6.3 ± 0.1 ng/cm². With a competitive format, hybridization reaction was carried out via immersing the DNA biosensor into a stirred hybridization solution containing different concentrations of the complementary ssDNA and constant concentration of nanosilver-labeled ssDNA, and then further binding with HRP. The adsorbed HRP amount on the probe surface decreased with the increment of the target ssDNA in the sample. The hybridization events were monitored by using differential pulse voltammetry (DPV) with the adsorbed HRP toward the reduction of H₂O₂. The reduction current from the enzyme-generated product was related to the number of target ssDNA molecules in the sample. A detection of 15 pmol/L for target ssDNA was obtained with the electrochemical DNA biosensor. Additionally, the developed approach can effectively discriminate complementary from non-complementary DNA sequence, suggesting that the similar enzyme-labeled DNA assay method hold great promises for sensitive electrochemical biosensor applications.     

Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry

A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.     

Giant magnetoresistive biochip for DNA detection and HPV genotyping

A giant magnetoresistive (GMR) biochip based on spin valve sensor array and magnetic nanoparticle labels was developed for inexpensive, sensitive and reliable DNA detection. The DNA targets detected in this experiment were PCR products amplified from Human Papillomavirus (HPV) plasmids. The concentrations of the target DNA after PCR were around 10nM in most cases, but concentrations of 10pM were also detectable, which is demonstrated by experiments with synthetic DNA samples. A mild but highly specific surface chemistry was used for probe oligonucleotide immobilization. Double modulation technique was used for signal detection in order to reduce the 1/f noise in the sensor. Twelve assays were performed with an accuracy of approximately 90%. Magnetic signals were consistent with particle coverage data measured with Scanning Electron Microscopy (SEM). More recent research on microfluidics showed the potential of reducing the assay time below one hour. This is the first demonstration of magnetic DNA detection using plasmid-derived samples. This study provides a direct proof that GMR sensors can be used for biomedical applications.     

Nanoparticle based DNA biosensor for tuberculosis detection using thermophilic helicase-dependent isothermal amplification

The present study describes the development of a DNA based biosensor to detect Mycobacterium tuberculosis using thermophilic helicase-dependent isothermal amplification (tHDA) and dextrin coated gold nanoparticles (AuNPs) as electrochemical reporter. The biosensor is composed of gold nanoparticles (AuNPs) and amine-terminated magnetic particles (MPs) each functionalized with a different DNA probe that specifically hybridize with opposite ends of a fragment within the IS6110 gene, which is M. tuberculosis complex (MTC) specific. After hybridization, the formed complex (MP-target-AuNP) is magnetically separated from the solution and the AuNPs are electrochemically detected on a screen printed carbon electrode (SPCE) chip. The obtained detection limit is 0.01 ng/μl of isothermally amplified target (105 bp). This biosensor system can be potentially implemented in peripheral laboratories with the use of a portable, handheld potentiostat.     

A simple fluorescent biosensor for theophylline based on its RNA aptamer

Theophylline is a potent bronchodilator with a narrow therapeutic index. A simple fluorescent biosensor that detects clinically relevant theophylline concentrations has been developed using the well-characterized theophylline binding RNA aptamer. Hybridization of the RNA aptamer to a fluorescently labeled DNA strand (FL-DNA) yields a fluorescent RNA:DNA hybrid that is sensitive to theophylline. The biosensor retains the remarkable selectivity of the RNA aptamer for theophylline over caffeine and is sensitive to 0-2 muM theophylline, well below the clinically relevant concentration (5-20 mg/L or approximately 10-50 muM). Adding a dabcyl quenching dye to the 3'-terminus of the fluorescently labeled DNA strand yielded a dual-labeled DNA strand (FL-DNA-Q) and increased the dynamic range of this simple biosensor from 1.5-fold to 4-fold.     

Fluorescent bio-barcode DNA assay for the detection of Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis is one of the most frequently reported causes of foodborne illness. It is a major threat to the food safety chain and public health. A highly amplified bio-barcode DNA assay for the rapid detection of the insertion element (Iel) gene of Salmonella Enteritidis is reported in this paper. The biosensor transducer is composed of two nanoparticles: gold nanoparticles (Au-NPs) and magnetic nanoparticles (MNPs). The Au-NPs are coated with the target-specific DNA probe which can recognize the target gene, and fluorescein-labeled barcode DNA in a 1:100 probe-to-barcode ratio. The MNPs are coated with the 2nd target-specific DNA probe. After mixing the nanoparticles with the 1st target DNA, the sandwich structure (MNPs-2nd DNA probe/Target DNA/1st DNA probe-Au-NPs-barcode DNA) is formed. A magnetic field is applied to separate the sandwich from the unreacted materials. Then the bio-barcode DNA is released from the Au-NPs. Because the Au-NPs have a large number of barcode DNA per DNA probe binding event, there is substantial amplification. The released barcode DNA is measured by fluorescence. Using this technique, the detection limit of this bio-barcode DNA assay is as low as 2.15 x 10(-16)mol (or 1 ng/mL).     

DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection

The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis.     

GMR sensors: magnetoresistive behaviour optimization for biological detection by means of superparamagnetic nanoparticles

An immunomagnetic method for the selective and quantitative detection of biological species by means of a magnetoresistive biosensor and superparamagnetic particles has been optimized. In order to achieve this, a giant magnetoresistive [Co (5.10nm)/Cu (2.47 nm)](20) multilayer structure has been chosen as the sensitive material, showing a magnetoresistance of 3.60% at 215 Oe and a sensitivity up to 0.19 Ω/Oe between 145 Oe and 350 Oe. The outward gold surface of the sensor is biofunctionalized with a Self-Assembled Monolayer (SAM). In addition, three different types of magnetic labels have been tested. 2 μm diameter magnetic carriers (7.68 pg ferrite/particle) have shown the best response and they have induced a shift in the magnetoresistive hysteresis loops up to 9% at 175 Oe.     

A Simple Fluorescent Biosensor for Theophylline Based on its RNA Aptamer

Theophylline is a potent bronchodilator with a narrow therapeutic index. A simple fluorescent biosensor that detects clinically relevant theophylline concentrations has been developed using the well-characterized theophylline binding RNA aptamer. Hybridization of the RNA aptamer to a fluorescently labeled DNA strand (FL-DNA) yields a fluorescent RNA:DNA hybrid that is sensitive to theophylline. The biosensor retains the remarkable selectivity of the RNA aptamer for theophylline over caffeine and is sensitive to 0–2 μM theophylline, well below the clinically relevant concentration (5–20 mg/L or ～10–50 μM). Adding a dabcyl quenching dye to the 3′-terminus of the fluorescently labeled DNA strand yielded a dual-labeled DNA strand (FL-DNA-Q) and increased the dynamic range of this simple biosensor from 1.5-fold to 4-fold.     

Development of an oral biosensor for salivary amylase using a monodispersed silver for signal amplification

An amperometric biosensor for monitoring the level of protein amylase in human saliva is described. A novel design and the preparation of amylase antibodies and antigens, essential for the development of the biosensor, are reported. The biosensor sensing elements comprise a layer of salivary antibody (or antigen) self-assembled onto Au-electrode via covalent attachment. Molecular recognition between the immobilized antibody and the salivary amylase proteins was monitored via an electroactive indicator (e.g., K(3)Fe(CN)(6)) or a monodispersed silver layer present in solution or electrochemically deposited onto the solid electrode. This electroactive indicator was oxidized or reduced and the resulting current change provided the analytical information about the concentration of the salivary proteins. The limit of detection of 1.57 pg/ml was obtained, in comparison to detection limits of 4.95 pg/ml obtained using potassium ferrocyanide as the redox probe and 10 ng/ml obtained using enzyme-linked immunosorbent assay. Cross-reactivity was tested against cystatin antibodies and was found to be less than 2.26%.     

The BARC biosensor applied to the detection of biological warfare agents

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.     

Electrogenerated chemiluminescence detection of adenosine based on triplex DNA biosensor

A novel electrogenerated chemiluminescence (ECL) biosensor based on the construction of triplex DNA for the detection of adenosine was designed. The ECL biosensor employs an aptamer as a molecular recognition element, and quenches ECL of tris(2,2'-bipyridine) ruthenium (Ru(bpy)(3)(2+)) by ferrocenemonocarboxylic acid (FcA). Through self-assembly technology, the ECL probe of thiolated hairpin adenosine aptamer tagged was self-assembled onto the surface of a gold electrode with an ECL signal producer Ru(bpy)(3)(2+) derivative (Ru-DNA-1). The adenosine aptamer, including a section of triplex characteristic chain, formatted triplex DNA with two other DNAs (DNA-2, Fc-DNA-3) in the presence of triplex DNA binder coralyne chloride (CORA). Fc-DNA-3 was tagged with an ECL quencher ferrocenemonocarboxylic acid (FcA), a quenching probe. In the presence of adenosine, the aptamer sequence (Ru-DNA-1) prefers to form the aptamer-adenosine complex with hairpin configuration and the switch of the DNA-1 occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of a quenching probe. Meanwhile, a control experiment was performed; the ECL-duplex biosensor was designed to detect adenosine. The detection limits were 2.7×10(-10) mol L(-1) and 2.3×10(-9) mol L(-1) for the ECL-triplex DNA biosensor and ECL-duplex DNA biosensor, respectively, which demonstrated that the ECL-triplex DNA biosensor improved the sensitivity and specificity greatly.     

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor.

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.