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The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody–aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated. info:eu-repo/semantics/publishedVersion

Aptamer-based assays and sensors are garnering increasing interest as alternatives to antibodies, particularly due to their increased flexibility for implementation in alternative assay formats, as they can be employed in assays designed for nucleic acids, such as molecular aptamer beacons or aptamer detection combined with amplification. In this work, we took advantage of the inherent nucleic acid nature of aptamers to enhance sensitivity in a rapid and facile assay format. An aptamer selected against the anaphylactic allergen β-conglutin was used to demonstrate the proof of concept. The aptamer was generated by using biotinylated dUTPs, and the affinity of the modified aptamer as compared to the unmodified aptamer was determined by using surface plasmon resonance to calculate the dissociation constant (KD), and no significant improvement in affinity due to the incorporation of the hydrophobic biotin was observed. The modified aptamer was then applied in a colorimetric competitive enzyme-linked oligonucleotide assay, where β-conglutin was immobilized on the wells of a microtiter plate, competing with β-conglutin free in solution for the binding to the aptamer. The limit of detection achieved was 68 pM, demonstrating an improvement in detection limit of three orders of magnitude as compared with the aptamer simply modified with a terminal biotin label. The concept was then exploited by using electrochemical detection and screen-printed electrodes where detection limits of 326 fM and 7.89 fM were obtained with carbon and gold electrodes, respectively. The assay format is generic in nature and can be applied to all aptamers, facilitating an easy and cost-effective means to achieve lower detection limits.

A 153-mer target DNA was amplified using ethynyl ferrocene dATP and a tailed forward primer resulting in a duplex with a single-stranded DNA tail for hybridization to a surface-tethered probe. A thiolated probe containing the sequence complementary to the tail as well as a 15 polythimine vertical spacer with a (CH

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, &beta, conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5&prime, or 3&prime, end or at both ends. The TT-tail at the 5&prime, end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

The use of lateral flow assays exploiting antibodies is well established in different fields due to their advantages, which include low cost, ease of production and rapid response, with the only required end-user intervention being sample addition. In recent years, aptamer-based lateral flow assays are garnering increasing interest offering a highly cost-effective and more flexible alternative to antibodies. In this review, an overview of the aptamer-based lateral flow assays developed to date is provided, highlighting the advantages of using aptamers and their ability to be incorporated into formats not possible with antibodies.

A simple, rapid and potentiostat-free method for grafting of close to monolayers of diazonium salts to anchor DNA probes for application in highly sensitive electrochemical genosensing is presented. Zn-mediated grafting is demonstrated to produce a highly organised surface facilitating enhanced sensitivity and a notably reduced detection limit as compared to electrochemical grafting. Keywords: Diazonium salt, Surface grafting, Genosensor, Amperometry

Carbon is a highly adaptable family of materials and is one of the most chemically stable materials known, providing a remarkable platform for the development of tunable molecular interfaces. Herein, we report a two-step process for the electrochemical hydrogenation of glassy carbon followed by either chemical or electrochemical chlorination to provide a highly reactive surface for further functionalization. The carbon surface at each stage of the process is characterized by AFM, SEM, Raman, attenuated total reflectance (ATR) FTIR, X-ray photoelectron spectroscopy (XPS), and electroanalytical techniques. Electrochemical chlorination of hydrogen-terminated surfaces is achieved in just 5 min at room temperature with hydrochloric acid, and chemical chlorination is performed with phosphorus pentachloride at 50 °C over a three-hour period. A more controlled and uniform surface is obtained using the electrochemical approach, as chemical chlorination is observed to damage the glassy carbon surface. A ferrocene-labeled alkylthiol is used as a model system to demonstrate the genericity and potential application of the highly reactive chlorinated surface formed, and the methodology is optimized. This process is then applied to thiolated DNA, and the functionality of the immobilized DNA probe is demonstrated. XPS reveals the covalent bond formed to be a C-S bond. The thermal stability of the thiolated molecules anchored on the glassy carbon is evaluated, and is found to be far superior to that on gold surfaces. This is the first report on the electrochemical hydrogenation and electrochemical chlorination of a glassy carbon surface, and this facile process can be applied to the highly stable functionalization of carbon surfaces with a plethora of diverse molecules, finding widespread applications.

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

Lupin has recently been added to the list of allergens requiring mandatory advisory labeling on foodstuffs sold in the European Union, and since December 2008, all products containing even trace amounts of lupin must be labeled correctly. Lupin globulins consist of two major globulins called α-conglutin (11S and "legumin-like") and β-conglutin (7S and "vicilin-like") and another additional two globulins, γ-conglutin and δ-conglutin, which are present in lower amounts. We report on a methodology to facilitate the extraction of each of these proteins using centrifugation and isolation by anion-exchange chromatography followed by size-exclusion chromatography. The isolated subunits were characterized using reducing and non-reducing polyacrylamide gel electrophoresis, western blotting, and peptide mass fingerprinting, all of which revealed that the individual protein subunits are highly pure and can be used as immunogens for the production of antibodies specific for each of the conglutin fractions, as well as standards, and the extraction protocol can be used for the selective extraction of each of the subunits from foodstuffs, thus facilitating a highly accurate determination of the lupin concentration. Furthermore, the subunits can be used to elucidate information regarding the toxicity of each of the subunits, by looking at their interaction with the IgE antibodies found in the serum of individuals allergic to lupin, providing critical information for the definition of the requirements of analytical assays for the detection of lupin in foodstuffs.

The objective of this work is to explore approaches to enhance electrochemical signals through sequential deposition and capping of gold particles. Gold nanoparticles are electrodeposited from KAuCl(4) solution under potentiostatic conditions on glassy carbon substrates. The number density of the nanoparticles is increased by multiple deposition steps. To prevent secondary nucleation processes, the nanoparticles are isolated after each potentiostatic deposition step by self-assembled monolayers (SAMs) of decanethiol or mercaptoethanol. The increasing number of particles during five deposition/protection rounds is monitored by assembling electroactive SAMs using a ferrocene-labeled alkanethiol. A precise estimation of the surface area of the gold nanoparticles by formation of an oxide layer on gold is difficult due to oxidation of the glassy carbon surface. As an alternative approach, the charge flow of the electroactive SAM is used for surface measurement of the gold surface area. A sixfold increase in the redox signal in comparison to a bulk gold surface is observed, and this increase in redox signal is particularly notable given that the surface area of the deposited nanoparticles is only a fraction of the bulk gold surface. After five rounds of deposition there is a gold loading of 1.94 mug cm(-2) of the deposited nanoparticles as compared to 23.68 mug cm(-2) for the bulk gold surface. Remarkably, however, the surface coverage of the ferrocene alkanethiol on the bulk material is only 10 % of that achieved on the deposited nanoparticles. This enhancement in signal of the nanoparticle-modified surface in comparison to bulk gold is thus demonstrated not to be attributable to an increase in surface area, but rather to the inherent properties of the surface atoms of the nanoparticles, which are more reactive than the surface atoms of the bulk material.

The vast majority of reports of self-assembled monolayers (SAMs) on metals focus on the use of gold. However, other metals, such as palladium, platinum, and silver offer advantages over gold as a substrate. In this work, palladium is electrochemically deposited from PdCl2 solutions on glassy carbon electrodes to form a substrate for alkanethiol SAMs. The conditions for deposition are optimized with respect to the electrolyte, pH, and electrochemical parameters. The palladium surfaces have been characterized by scanning electron microscopy (SEM) and the surface roughness has been estimated by chronocoulometry. SAMs of alkane thiols have been formed on the palladium surfaces, and their ability to suppress a Faradaic process is used as an indication for palladium coverage on the glassy carbon. The morphology of the Pd deposit as characterized by SEM and the blocking behavior of the SAM formed on deposited Pd delivers a consistent picture of the Pd surface. It has been clearly demonstrated that, via selection of experimental conditions for the electrochemical deposition, the morphology of the palladium surface and its ability to support SAMs can be controlled. The work will be applied to create a mixed monolayer of metals, which can subsequently be used to create a mixed SAM of a biocomponent and an alkanethiol for biosensing applications.

Potential-controlled partial reductive desorption of a self-assembled monolayer of mercaptopropionic acid (MPA) formed on polycrystalline gold electrodes is used to expose subdomains of bare gold to the electrolyte solution. Two sets of cathodic waves are observed in the reduction scan with MPA self-assembled on a polycrystalline gold electrode. The origin of the two waves is ambiguous but there are indications that the waves are correlated with reductive desorption from the (111) and then simultaneously (100) and (110) index faces of a polycrystalline gold electrode. Consecutive reduction scans with reversing the potential direction after the first peak (but before the onset of the second wave) results in disappearance of the first wave. The exposed domains are then blocked by an assembling process of longer chain alkanethiols to create a mixed self-assembled monolayer on polycrystalline gold electrodes. Desorption of the remaining MPA creates a partially blocked electrode and the blocking behavior towards hexacyanoferrate(III) is analyzed using the theory of partially blocked electrodes and indicates an array of interacting centers. The approach of partial reductive desorption may be exploited for use in biosensing applications where the exposed gold domains could be used for anchoring of DNA probes.

Direct detection of PCR product via hybridisation assay, would facilitate the development of rapid tools for genetic analysis. Here, a PCR primer designed to generate a PCR amplicon tagged with single stranded DNA tails at each end of the duplex, which can be used for direct hybridisation with a surface immobilised probe and an enzyme labelled reporter probe is presented. Four modified sequence specific primers (SSP) pairs were designed for the selective amplification of coeliac disease associated alleles (DQA1*05, DQB1*02, DQB1*03:02 alleles), and human growth hormone (positive control). Multiplex PCR products were electrochemically detected in less than 5 min at 37 °C via direct hybridisation to short probes immobilised on individual electrodes of a genosensor array, and subsequent hybridisation to an enzyme labelled reporter probe. The developed electrochemical genosensor array exploiting the modified primers for the direct detection of PCR products was applied to the genotyping of real patient samples.

A simple and sensitive method for the electrocatalytic detection of NADH on a glassy carbon electrode modified with electrografted o-aminophenol film (o-AP) is presented. The modification of a glassy carbon electrode surface with an o-AP film was achieved by electrochemical reduction of the corresponding, in situ generated nitrophenyl diazonium cation. The functionalized electrode shows an efficient electrocatalytic activity towards the oxidation of NADH with activation overpotential, which is ca. 350 mV lower than that at the bare electrode. The stability and the electrocatalytic activity of the modified electrode have been critically addressed. The formation of an intermediate charge transfer complex is proposed for the charge transfer reaction between NADH and adsorbed o-AP. The second-order rate constant for electrocatalytic oxidation of NADH, kobs, and the apparent Michaelis–Menten constant KM, at pH 7.0 were evaluated with rotating disk electrode (RDE) experiments, using the Koutecky–Levich approach. Using the o-AP-GC electrode, at an applied potential of +150 mV (vs. Ag–AgCl) with amperometric detection of NADH, a calibration range from 7.5 × 10−7 to 2.5 × 10−6 with a detection limit of 1.5 × 10−7 M was obtained, and excellent reproducibility was demonstrated with an RSD% = 2.1, n = 10 using a concentration of 1.0 × 10−6 M NADH. Keywords: Electrocatalytic oxidation, NADH, Diazonium salt, Surface modification, NADH sensor, Nanostructured selectively catalytic surface

Here, we report a simple and extremely effective method to chemically grafting of o -aminophenol film ( o -AP) onto glassy carbon (GC) electrode by the electrochemical reduction of the corresponding nitrophenyl diazonium salt in acidic aqueous solution for the electrocatalytic detection of hydrazine. The covalently attached o -AP enabled hydrazine to be catalytically oxidized at a greatly reduced overpotential and in a wide operational pH range. A quinoneimine structure has been proposed as the electrocatalytically active species. The kinetics of the reaction between the o -AP mediator and hydrazine has been characterized using cyclic voltammetry, chronoamperometry, and chronocoulometry and rotating disk electrode voltammetry. The o -AP modified electrode as an amperometric sensor has been characterized. The catalytic currents were proportional to the concentration of hydrazine giving rise to calibration curves characterized by two linear segments. The linear segment over the concentration range of 2.0–20.0 μM could be used with analytical purposes to determination of hydrazine with a detection limit of 0.5 μM and a sensitivity of 0.016 μA/μM. The precision of amperometry was found to be 1.7% for replicate determinations ( n = 10) of a 10 μM solution of hydrazine. The resulting modified electrode retains its initial response for at least one month if stored dry in air.

Layer-by-Layer (LbL) assemblies are multi-modular constructions created on solid substrate mainly via electrostatic interactions between polyanions and polycations. The LbL approach can be used for the inter-layer immobilization of biomolecules with enhanced properties for biocatalytic and immunosensing applications, and to create nanoarchitectures for drug encapsulation and release systems. This LbL approach is generic and provides a strategy to rationally design the properties of immobilized films. The following review reports on the LbL state-of-the-art, characterization techniques, applications in biosensing, and nanoarchitectures.

Self-assembly of a ferrocene-appended polymer bearing an antigen fragment and lactate oxidase on a cyclodextrin-modified surface provides a highly sensitive, easy-to-operate and self-sufficient immunosensor.

Tetrabutyl ammonium (TBA) salts of organotin derivatives of polyoxometalates (TBA)5[SiW11O39{Sn(CH2)2COOH}] (1), (TBA)4[SiW11O39{Sn(CH2)2CO}] (2), (TBA)5[SiW11O39{Sn(CH2)2CONH(CH2)3N3}] (3), TBA5[SiW11O39{Sn(CH2)2CONH(CH2)3(N3C2H)C10H9Fe}] (4) and an ammonium salt (NH4)5[SiW11O39{Sn(CH2)2CONH(CH2)3N3}](3′) have been synthesized for the first time by adapting the organic functionalization strategies developed earlier for phosphotungstates. The products were characterized using FTIR, NMR, ESI MS, and electrochemical techniques. The Keggin silicotungstate is more nucleophilic than the Keggin phosphotungstate and displays reactivity previously known only for Dawson phosphotungstates. The methodology of CuAAC “click” reaction for silico- and phosphotungstates allows the coupling by a common protocol of diverse POMs with different redox properties, which is potentially useful for bioelectroanalytical applications with redox labels.

A bifunctionalised carboxymethyl-cellulose polymer bearing adamantane units and an antigenic fragment forms a highly stable interfacial complex with a βCD-containing surface. This allows the highly sensitive detection of antibodies using an amperometric immunosensor.

Single-stranded guanosine (G)-rich oligonucleotides have a propensity to form quadruplex structures stabilized by 4-G quartets. The four guanines of such a quartet array in a square planar conWguration, each G interacting with the adjacent one via two hydrogen bonds and behaving as H acceptor/donor of what is known as a Hoogsteen basepair. These structures are believed to exist only in the presence of certain cations, described to stabilize the quadruplex, presumably by compensating the intermolecular repulsion due to the negative charges of the nucleic acid [1–3]. Proteins able to bind such structures under physiological conditions have been described [3,4], and two of them have even been suggested to promote quadruplex formation [5,6]. The fact that some quadruplex-forming sequences have been described to inhibit proliferation of viruses or cancer cells is being exploited for its potential therapeutic application [1]. The thrombin-binding aptamer (TBA)1 was the Wrst in vitro-selected oligonucleotide targeted toward a protein that has no known physiological binding to nucleic acids. The TBA is known to be in equilibrium between a random and a quadruplex conformation when dissolved in aqueous solution [7]. This quadruplex structure contains two G(syn)-G(anti)-G(syn)-G(anti) quartets and three lateral loops, in what is usually referred to as a “chair structure” [8–11]. The presence of K+ in the medium was believed to be necessary to shift the equilibrium toward the quadruplex conformation, subsequently

In this work, we describe a novel route for the immobilisation of an electron transfer mediator on electrode surfaces based on the interfacial complexation of a bifunctionalised carboxymethylcellulose (CMC) polymer backbone bearing ferrocene units and a DNA probe on a cyclodextrin-functionalised surface. The interfacial self-assembly has been studied using surface plasmon resonance and electrochemical techniques and the applicability of the modified surface for the construction of an amperometric genosensor was explored for the detection of a celiac disease associated allele. The supramolecular strategy simplifies the operation of the biosensor, only requiring the addition of an enzyme substrate, and the proximity of the mediator to the electrode surface greatly improves the detection limits attained (10 pM) with respect to a similar supramolecular system based on electrochemically inactive adamantane/CD inclusion complexes (80 pM) and requiring addition of the mediator in solution. In addition, the use of the hydrophilic CMC backbone contributes to the elimination of non-specific interactions and to an optimal spacing of the immobilised DNA probes.

Coeliac disease is an inflammatory disease of the upper small intestine and results from gluten ingestion in genetically susceptible individuals, and is the only life-long nutrient-induced enteropathy. The only treatment is a strict gluten-free diet and the longer the individual fails to adhere to this diet, the greater the chance of developing malnutrition and other complications. The existence of reliable gluten free food is crucial to the well-being of the population. Here we report on a microfluorimeter device for the in situ detection of gliadin in foodstuffs, which could be used for a rapid control of raw materials in food processing, as well as for process control of gliadin contamination. The microfluorimeter is based on a reflector that is used inside a microfluidic chip, exploiting various strategically placed reflective or totally metallised mirrors for efficient collection of the fluorescent light emitted in a large solid angle. The chip is capable of executing five assays in parallel and has been demonstrated to possess detection sensitivity applicable to fluoroimmunoassays. Various immunoassay formats exploiting fluorescence detection, using enzyme/fluorophore labels were developed and compared in terms of sensitivity, ease of assay, assay time and compatibility with buffer used to extract gliadin from raw and cooked foodstuffs, with the best performance observed with an indirect competition assay using a fluorophore-labelled anti-mouse antibody. This assay was exploited within the microfluorimeter device, and a very low detection limit of 4.1 ng/mL was obtained. The system was observed to be highly reproducible, with an RSD of 5.9%, for a concentration of 50 ng/mL of gliadin applied to each of the five channels of the microfluorimeter. Biofunctionalised disposable strips incorporated into the microfluorimeter were subjected to accelerated Arrhenius thermal stability studies and it was demonstrated that strips pre-coated with gliadin could be stored for approximately 2 years at 4 degrees C, with no discernable loss in sensitivity or detectability of the assay. Finally, the microfluorimeter was applied to the analysis of commercial gluten-free food samples, and an excellent correlation with routine ELISA measurements was obtained. The developed microfluorimeter should find widespread application for on-site execution of fluoroimmunoassays.

We report the first use of an electrochemical aptasensor for selective potassium recognition, based on a conformational change, affording an electric signal transduced electrochemically by square wave voltammetry or electrochemical impedance spectroscopy.

Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery and reamplification. Aptamers, first reported in 1990, are attracting interest in the areas of therapeutics and diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors. The properties of aptamers, their applicability to biosensor technology, current research and future prospects are addressed in this short review.