Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection.

Highly sensitive testing of nucleic acids is essential to improve the detection of pathogens, which pose a major threat for public health worldwide. Currently available molecular assays, mainly based on PCR, have a limited utility in point-of-need control or resource-limited settings. Consequently, there is a strong interest in developing cost-effective, robust, and portable platforms for early detection of these harmful microorganisms. Since its description in 2004, isothermal helicase-dependent amplification (HDA) has been successfully applied in the development of novel molecular-based technologies for rapid, sensitive, and selective detection of viruses and bacteria. In this review, we highlight relevant analytical systems using this simple nucleic acid amplification methodology that takes place at a constant temperature and that is readily compatible with microfluidic technologies. Different strategies for monitoring HDA amplification products are described. In addition, we present technological advances for integrating sample preparation, HDA amplification, and detection. Future perspectives and challenges toward point-of-need use not only for clinical diagnosis but also in food safety testing and environmental monitoring are also discussed. Graphical Abstract Expanding the analytical toolbox for the detection of DNA sequences specific of pathogens with isothermal helicase dependent amplification (HDA).

A Quantitative PCR-Electrochemical Genosensor Test for the Screening of Biotech Crops.

The design of screening methods for the detection of genetically modified organisms (GMOs) in food would improve the efficiency in their control. We report here a PCR amplification method combined with a sequence-specific electrochemical genosensor for the quantification of a DNA sequence characteristic of the 35S promoter derived from the cauliflower mosaic virus (CaMV). Specifically, we employ a genosensor constructed by chemisorption of a thiolated capture probe and p-aminothiophenol gold surfaces to entrap on the sensing layer the unpurified PCR amplicons, together with a signaling probe labeled with fluorescein. The proposed test allows for the determination of a transgene copy number in both hemizygous (maize MON810 trait) and homozygous (soybean GTS40-3-2) transformed plants, and exhibits a limit of quantification of at least 0.25% for both kinds of GMO lines.

Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay.

Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay.

Enzyme immunoassays are currently the methods of choice for gluten control in foods labelled as gluten free, providing a mechanism for assessing food safety for consumption by coeliac and other allergic patients. However, their limitations, many of them associated to the reactivity of the different antibodies used and their degree of specificity, have prevented the establishment of a standardised method of analysis. We explore new methods for quantitatively determining gluten content in foods based on the use of two recently described aptamers, raised against a 33-mer peptide recognised as the immunodominant fragment from α2-gliadin. The assays use the target peptide immobilised onto streptavidin-coated magnetic beads in combination with a limited amount of biotin-aptamer in a competitive format, followed by streptavidin-peroxidase labelling of the aptamer that remains bound to the magnetic beads. The enzyme activity onto the beads, measured by chronoamperometry in disposable screen-printed electrodes, is inversely related to the target concentration in the test solution. We find that while the assay using the aptamer with the highest affinity towards the target (Gli 4) achieves low detection limits (~0.5 ppm) and excellent analytical performance, when challenged in samples containing the intact protein, gliadin, it fails in detecting the peptide in solution. This problem is circumvented by employing another aptamer (Gli 1), the most abundant one in the SELEX pool, as a receptor. The proposed assays allow the convenient detection of the allergen in different kinds of food samples, including heat-treated and hydrolysed ones. The obtained results correlate with those of commercially available antibody-based assays, providing an alternative for ensuring the safety and quality of nominally gluten-free foods. Graphical Abstract Electrochemical magnetoassay for gluten determination using biotin-aptamers as receptors.

Aptamer binding to celiac disease-triggering hydrophobic proteins: a sensitive gluten detection approach.

Celiac disease represents a significant public health problem in large parts of the world. A major hurdle in the effective management of the disease by celiac sufferers is the sensitivity of the current available methods for assessing gluten contents in food. In response, we report a highly sensitive approach for gluten analysis using aptamers as specific receptors. Gliadins, a fraction of gluten proteins, are the main constituent responsible for triggering the disease. However, they are highly hydrophobic and large molecules, regarded as difficult targets for in vitro evolution of aptamers without nucleobase modification. We describe the successful selection of aptamers for these water insoluble prolamins that was achieved choosing the immunodominant apolar peptide from α2-gliadin as a target for selection. All aptamers evolved are able to bind the target in its native environment within the natural protein. The best nonprotein receptor is the basis for an electrochemical competitive enzyme-linked assay on magnetic particles, which allows the measurement of as low as 0.5 ppb of gliadin standard (0.5 ppm of gluten). Reference immunoassay for detecting the same target has a limit of detection of 3 ppm, 6 times less sensitive than this method. Importantly, it also displays high specificity, detecting the other three prolamins toxic for celiac patients and not showing cross-reactivity to nontoxic proteins such as maize, soya, and rice. These features make the proposed method a valuable tool for gluten detection in foods.

Aptamer-based analysis: a promising alternative for food safety control.

Ensuring food safety is nowadays a top priority of authorities and professional players in the food supply chain. One of the key challenges to determine the safety of food and guarantee a high level of consumer protection is the availability of fast, sensitive and reliable analytical methods to identify specific hazards associated to food before they become a health problem. The limitations of existing methods have encouraged the development of new technologies, among them biosensors. Success in biosensor design depends largely on the development of novel receptors with enhanced affinity to the target, while being stable and economical. Aptamers fulfill these characteristics, and thus have surfaced as promising alternatives to natural receptors. This Review describes analytical strategies developed so far using aptamers for the control of pathogens, allergens, adulterants, toxins and other forbidden contaminants to ensure food safety. The main progresses to date are presented, highlighting potential prospects for the future.

Thioaromatic DNA monolayers for target-amplification-free electrochemical sensing of environmental pathogenic bacteria.

Genosensing technology has mostly based on mixed self-assembled monolayers (SAMs) of thiol-modified oligonucleotides and alkanethiols on gold surfaces. However, the typical backfilling approach, which incorporates the alkanethiol in a second step, gives rise to a heterogeneous distribution of oligonucleotide probes on the surface, negatively affecting to both hybridization efficiency and surface stability. Despite aromatic thiols present a remarkably different behavior from alkanethiols, with higher rigidity and stronger intermolecular interactions, they have been scarcely explored for the fabrication of DNA sensing platforms. We have investigated different approaches involving SAMs of aromatic thiols, namely p-mercaptobenzoic acid (p-MBA) and p-aminothiophenol (p-ATP), to yield DNA sensing layers for sequence-specific detection of target oligonucleotides. The studied monolayers were evaluated by DNA surface coverage and further information was obtained by determining their functionality in a sandwich hybridization assay with enzymatic amplification of the electrochemical read-out. The insertion of thiol-oligonucleotides into p-ATP monolayers previously oxidized, and the covalent binding of amino-oligonucleotides to pure p-MBA monolayers give rise to increased storage stability and better analytical performance. The quantification of RNA from Legionella pneumophila cellular lysates was successfully performed, illustrating the usefulness of these sensing architectures for detecting pathogenic bacteria.

Computational predictions and experimental affinity distributions for a homovanillic acid molecularly imprinted polymer.

Density Functional Theory calculations have been used to select, among a set of chemicals traditionally used in the formulation of non-covalent molecularly imprinted polymers (MIPs), the best functional monomer and porogenic solvent for the construction of a recognition element for the dopamine metabolite homovanillic acid (HVA). Theoretical predictions were confirmed through batch binding assays and voltammetric detection. The computational method predicts that trifluoromethacrylic acid and toluene are the monomer and solvent rendering the highest stabilization energy for the pre-polymerization adducts. HVA-MIP prepared using this formulation gives rise to a binding isotherm that is accurately modelled by the Freundlich isotherm. The binding properties of this polymer were estimated using affinity distribution analysis. An apparent number of sites of 13 micromol g(-1) with an average affinity constant of 2 x 10(4) M(-1) was obtained in the concentration window studied.

Amplified label-free electrocatalytic detection of DNA in the presence of calcium ions.

A new label-free electrocatalytic method for the detection of DNA, is presented, in which DNA is the catalyst. The method takes advantage of the catalytic properties of the electrooxidised adenines within DNA toward the oxidation of NADH. This catalytic event results in an enhancement in the oxidation current of the electrooxidised adenines within DNA. Further improvement in this analytical signal is achieved in the presence of Ca(2+) ions. Parameters affecting the electrocatalytic current, such as pH or concentration of Ca(2+) ions have been investigated and optimised. Finally, the analytical features of the developed method are obtained. This method constitutes a more sensitive and reproducible alternative to other methods that use the oxidation current of the electrooxidised adenines without coupling to any catalytic event. A limit of detection of 33 fmol deoxyadenylic acid icosanucleotide (dA)(20), is obtained without labels.

Harnessing Aptamers to Overcome Challenges in Gluten Detection.

Celiac disease is a lifelong autoimmune disorder triggered by foods containing gluten, the storage protein in wheat, rye, and barley. The rapidly escalating number of patients diagnosed with this disease poses a great challenge to both food industry and authorities to guarantee food safety for all. Therefore, intensive efforts are being made to establish minimal disease-eliciting doses of gluten and consequently to improve gluten-free labeling. These efforts depend to a high degree on the availability of methods capable of detecting the protein in food samples at levels as low as possible. Current analytical approaches rely on the use of antibodies as selective recognition elements. With limited sensitivity, these methods exhibit some deficiencies that compromise the accuracy of the obtained results. Aptamers provide an ideal alternative for designing biosensors for fast and selective measurement of gluten in foods. This article highlights the challenges in gluten detection, the current status of the use of aptamers for solving this problem, and what remains to be done to move these systems into commercial applications.

Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection.

A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65°C, providing 3×10(6) amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available.

Strongly structured DNA sequences as targets for genosensing: sensing phase design and coupling to PCR amplification for a highly specific 33-mer gliadin DNA fragment.

Electrochemical genosensors are becoming cost-effective miniaturizable alternatives to real-time PCR (RT-PCR) methods for the detection of sequence-specific DNA fragments. We report on the rapid detection of PCR amplicons without the need of purification or strand separation. A challenging target sequence for both PCR amplification and electrochemical detection allowed us to address some difficulties associated to hybridization on electrode surfaces. The target was a highly specific oligonucleotide sequence of wheat encoding the most immunogenic peptide of gliadin that triggers the immune response of celiac disease (CD), the 33-mer. With a sandwich assay format and a rational design of the capture and tagged-signaling probes the problems posed by the strong secondary structure of the target and complementary probes were alleviated. Using a binary self-assembled monolayer and enzymatic amplification, a limit of detection of 0.3 nM was obtained. The genosensor did not respond to other gluten-containing cereals such as rye and barley. Coupling to PCR to analyze wheat flour samples required tailoring both the capture and signaling probes. This is the first time that deleterious steric hindrance from long single-stranded regions adjacent to the electrode surface is reported for relatively short amplicons (less than 200 bp). The importance of the location of the recognition site within the DNA sequence is discussed. Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour.

PCR-coupled electrochemical sensing of Legionella pneumophila.

Human infections with Legionella pneumophila represent a public health problem. Current culture assays for surveillance and control of L. pneumophila in water are time-consuming and limited by the sensitivity, especially when samples also contain microorganisms that inhibit Legionella growth. In this work, an electrochemical method, different from real-time polymerase chain reaction (PCR) approaches, for semiquantitative evaluation of L. pneumophila is presented. A PCR assay targeting the 16S-rRNA gene of L. pneumophila giving rise to a 95-mer amplicon was established. Amplicons were hybridized to a biotin-labeled reporter sequence and then to a thiolated stem-loop structure immobilized onto gold electrodes as a reporter molecule with 1-naphthyl phosphate as a substrate. 1-Naphthol enzymatically generated was determined by differential pulse voltammetry (DPV). For a constant number of amplification cycles, results show that the voltammetric signal is related to the number of copies in the sample thus achieving a useful semiquantitative estimation of L. pneumophila. After 40 cycles of PCR amplification this methodology has a limit of detection of 10 genomes, allowing the reliable detection of 10(2) genomes of L. pneumophila as well as distinguishing 10(3) and 10(4) genomes of the pathogen, values related to corrective actions in water systems in buildings, in accordance with the legislation currently in force.

SPR sensing of small molecules with modified RNA aptamers: detection of neomycin B.

We have studied how the modification of the RNA aptamer evolved against neomycin B at 2' position of ribose with a methyl group influences the affinity of the interaction. Using surface plasmon resonance (SPR) and faradaic impedance spectroscopy (FIS) an affinity constant in the muM range was calculated. The results showed that the modification of the aptamer does not significantly alter the affinity of the aptamer for the antibiotic. This finding opens up the possibility of designing modified RNA aptamers resistant to endonucleases without variation of the analytical features. In addition to this, we propose a competitive assay for the detection of neomycin B using SPR as a transduction technique. A range of quantification between 10 nM and 100 microM was obtained, which shows the feasibility of detecting small molecules using aptamers with high sensitivity.

Hairpin-DNA probe for enzyme-amplified electrochemical detection of Legionella pneumophila.

An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 muM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.

5-Hydroxytryptophan as a precursor of a catalyst for the oxidation of NADH.

Following oxidation of 5-hydroxytryptophan (5-HTPP) at a pyrolytic graphite electrode at pH 7.5, two quasi-reversible redox couples emerge at -0.170 and +0.032 V, respectively, due to oxidation products strongly adsorbed to the electrode surface. These redox processes have been electrochemically and kinetically characterized in terms of the dependence of the formal potential (E degrees ') with pH, variation of the current density with scan rate, operational stability, and electron-transfer rate constant (k(s)). The wave centered at +0.032 V could mediate the oxidation of NADH, exhibiting a strong and persistent electrocatalytic response. A quinone-imine structure has been proposed as the electrocatalytically active species. The kinetics of the reaction between the mediator and NADH has been characterized via rotating disk electrode voltammetry, and it has been found that the rate constant for the reaction is dependent on the solution concentration of NADH. 5-HTPP modified electrodes could be employed in the amperometric detection of NADH with a limit of detection in the nanomolar range. Moreover, 5-HTPP modified electrodes retain their electrocatalytic activity for at least one week. The potential application of these electrodes to amperometric biosensor is demonstrated.

Flavin adenine dinucleotide as precursor for NADH electrocatalyst.

The generation of a new electrocatalytic system for NADH after oxidizing flavin adenine dinucleotide (FAD) is shown. The oxidation is performed in alkaline medium until +1.4 V (Ag/AgCl) at graphite electrodes. The catalytic activity is ascribed to the electrooxidized moiety of FAD and not to quinone surface groups. A comparison between this catalyst and that attributed to poly(FAD) (Karyakin, A. A.; Ivanova Y. N.; Revunova, K. V.; Karyakina, E. E. Anal. Chem. 2004, 76, 2004-2009.) is presented. It is concluded that the surface quinone groups generated during the strong anodization of the electrode in acidic medium at 2-2.5 V and not the poly(FAD) are responsible for the catalytic activity described in the above mentioned work.

Computational approach to the rational design of molecularly imprinted polymers for voltammetric sensing of homovanillic acid.

A methodology based on density functional theory calculations for the design of molecularly imprinted polymers (MIPs) is described. The method allows the rational choice of the most suitable monomer and polymerization solvent among a set of chemicals traditionally used in MIP formulations for the molecular imprinting of a given template. It is based on the comparison of the stabilization energies of the prepolymerization adducts between the template and different functional monomers. The effect of the polymerization solvent is included using the polarizable continuum model. A voltammetric sensor for homovanillic acid was constructed using different MIPs as recognition element, confirming that the solvent (toluene) and functional monomer (methacrylic acid) selected according to the theoretical predictions lead to the most efficient molecular recognition sensing phase. With the voltammetric sensor prepared using the MIP designed according to the theoretical predictions, a linear response for concentrations of homovanillic acid between 5 x 10(-8) and 1 x 10(-5) M can be obtained. The limit of detection is 7 x 10(-9) M. The selectivity obtained for homovanillic acid over other structurally related compounds buttresses the validity of this strategy of design.

Modified carbon paste electrodes for flow injection amperometric determination of isocitrate dehydrogenase activity in serum.

A carbon paste electrode modified with the adsorbed products of the electrochemical oxidation of adenosine triphosphate is described. The electrode was applied to the amperometric electrocatalytic detection of the reduced form of both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. The catalytic oxidation current shows a linear dependence on the concentration of the reduced form of nicotinamide adenine dinucleotide up to 1x10(-4)M, with a detection limit of 5x10(-9)M. Modified carbon paste electrodes were coated with an electrogenerated film of nonconducting poly(o-phenylenediamine) to obtain a stable amperometric response for at least 150h. In addition to static measurements, determination of both reduced cofactors was carried out in a flow injection analysis system with a thin-layer amperometric detection cell. The electrocatalytic monitoring of reduced nicotinamide adenine dinucleotide phosphate was applied to flow injection measurement of isocitrate dehydrogenase activity in serum. The results were in good agreement with those for the standard spectrophotometric test kit. The proposed method consumed less time and reagents and provided better precision than the standard method.