An electrochemical gene detection assay utilising T7 exonuclease activity on complementary probe–target oligonucleotide sequences

This communication describes the synthesis of an electrochemically active oligonucleotide probe and its application in sensing complementary oligonucleotides sequences using a T7 exonuclease enzyme. Target oligonucleotides are detected by hybridisation with a ferrocene labelled probe oligonucleotide followed by addition of T7 exonuclease. The T7 enzyme is a double strand specific exonuclease that removes the terminal 5′ nucleotide of the probe sequence. The 5′ nucleotide is attached to a ferrocene label, which is subsequently detected at an electrode using differential pulse voltammetry. Time and temperature resolved measurements were performed and an associated study using dual labelled fluorophore–quencher labelled probes was performed to confirm the validity of the electrochemical assay.     

M-DNA: a self-assembling molecular wire for nanoelectronics and biosensing.

M-DNA is a complex between divalent metal ions such as Zn2+ and duplex DNA which forms at pH 8.5. Unlike B-DNA, M-DNA does not bind ethidium so that M-DNA formation can be monitored conveniently by an ethidium fluorescence assay. M-DNA was shown to be a better conductor than B-DNA by fluorometric measurements of electron transport in donor-acceptor labelled duplexes; by direct conductivity measurements of M-DNA bound between gold electrodes and by cyclic voltammetric studies on ferrocene labelled duplexes attached to gold microelectrodes. As is the case with B-DNA, M-DNA can self-assemble into a variety of structures and is anticipated to find widespread use in nanoelectronics and biosensing.     

Electrochemical melting-curve analysis

Genotyping technologies need to tackle issues of cost-effectiveness, flexibility, and mutiplexability to meet the ever-increasing demands for clinical diagnostics, addressing the future medical paradigm. Here we report on a facile method for the rapid detection of mutations using electrochemical melting-curve analysis. The concept is based on the use of an immobilised probe hybridised to the mutant region of a ferrocene labelled amplicon. Following hybridisation, the temperature is ramped and the dissociation of the ferrocene labelled DNA from the electrode surface is monitored using differential pulse voltammetry. Using a model system consisting of short probe and target, the proposed approach was demonstrated to clearly discriminate between complementary and mismatch duplexes. The melting temperature of the surface confined DNA duplex was observed to be markedly lower than that obtained in solution, with melting temperatures of 38 and 59 °C, observed, respectively. The approach can be extended to array based melting-curve analysis, allowing the simultaneous detection of multiple mutations, as well as for genosensor design.     

Nanostructured DNA Microarrays for Dual SERS and Electrochemical Read‐out

We present a strategy to fabricate nanostructured microarrays ready to perform a dual read‐out, namely electrochemical (EC) as well as surface‐enhanced Raman spectroscopy (SERS) based detection of DNA hydridization. A polystyrene nanobeads monolayer assembly, obtained by means of a Langmuir Blodgett type technique, followed by electrochemical Au deposition, was employed to construct homogeneous nanostructures in the form of inverse‐opal nanovoids on a 32‐electrode Au microarray chip. Characterization of the obtained nanostructured electrodes of the array by means of cyclic voltammetry demonstrated high reproducibility of the surface modification process. The performance of the obtained array platform was investigated by modifying the microarray electrodes with three different oligonucleotide capture probes using a previously developed potential‐assisted surface modification protocol. Two ferrocene‐labeled target DNA sequences and one target RNA sequence with a Texas red label were detected electrochemically and via SERS, respectively.     

Quenching of the electrochemiluminescence of tris(2,2'-bipyridine)ruthenium(II) by ferrocene and its potential application to quantitative DNA detection

Efficient and stable quenching of electrochemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(II) by oxidizing ferrocene methanol (FcMeOH) at the electrode is reported. Bimolecular energy or electron transfer between Ru(bpy)(3)(2+*) and ferrocenium (Fc(+)), the oxidized species of Fc, along with suppression of radical reactions is suggested as the mechanism for quenching ECL. Fc shows more efficient quenching of ECL compared with the known quenchers phenol and 1,1-dimethyl-4,4'-bipyridine dication (MV(2+)). The ECL quenching rate constant was 5.6 x 10(10) M(-)(1) s(-)(1). Using Fc as a quencher label on a complementary DNA sequence, an intramolecular ECL quenching in hybridized oligonucleotide strands has been realized. With essentially complete quenching efficiency, this system has the potential for application to sequence-specific DNA detection.     

Purification of radioiodinated photoactivable glucagon by isocratic high-performance liquid chromatography

2-Nitro-4-azidophenylsulphenyl-glucagon, a specific photoaffinity label for glucagon receptors, was synthesized and radioactively labelled with 125I. The radio-labelled peptide was purified from the reaction mixture by high-performance liquid chromatography in one step by isocratic elution from a C18 column with 20.4% n-propanol in 10 mM phosphate buffer (pH 2.5) as eluent. This glucagon derivative can be used to attach a label specifically to the glucagon receptor. The binding ability of the photoaffinity derivative was tested on isolated intact rat hepatocytes. Compared with a Kd of 3 nM for unmodified monoiodinated glucagon, the Kd value of the photoaffinity labelled monoiodinated glucagon tracer was 7 nM.     

Towards a target label-free suboptimum oligonucleotide displacement-based detection system

A novel method for the future development of label-free DNA sensors is proposed here. The approach is based on the displacement of a labelled suboptimum mutated oligonucleotide hybridised with the immobilised biotin-capture probe. The target fully complementary to the biotin-capture probe can displace the labelled oligonucleotide causing a subsequent decrease of the signal that verifies the presence of the target. The decrease of signal was demonstrated to be proportional to the target concentration. A study of the hybridisation of mutated and complementary labelled oligonucleotides with an immobilised biotin-capture probe was carried out. Different kinetic and thermodynamic behaviour was observed for heterogeneous hybridisation of biotin-capture probe with complementary or suboptimum oligonucleotides. The displacement method evaluated colourimetrically achieved the objective of decreasing the response time from 1 h for direct hybridisation of 19-mer oligonucleotides in the direct enzyme-linked oligonucleotide assay (ELONA) to 5 min in the case of displacement detection in the micromolar concentration range. Figure The detection system is based on the displacement of suboptimum HRP-labelled mutated oligonucleotide by the fully complementary target     

A Ruthenium Based Organometallic Complex for Biosensing that is both a Stable Redox Label and a Homobifunctional Linker

A stable ruthenium‐based redox label, Ru(acetylacetonate)₂(bipyridine‐NH₂), has been synthesized with the target of circumventing the problem associated with the use of ferrocene for biosensing in solutions containing chloride ions. The redox species was shown to be highly stable with repeated cycling in biological buffers as well as being amenable to surface coupling reactions. To demonstrate the latter, the redox label was anchored onto a self‐assembled monolayer of 6‐mercaptohexanoic acid, using carbodiimide coupling, followed by binding of a pentapeptide to the redox label.     

Ultrasensitive detection based on an aptamer beacon electron transfer chain

The construction of an ultrasensitive, reagentless, target label free electrochemical aptamer sensor (aptasensor) for thrombin detection is described. The aptasensor is based on a chronoamperometric beacon system for biomolecular recognition. The ferrocene-labeled aptamer adopts a 3-D conformational change when interacts with thrombin. Thus the ferrocene label is approached to the microperoxise-11 (MP-11) attached on the electrode surface. The thrombin–aptamer interaction is detected via a microperoxidase mediated electron transfer between the ferrocene and the surface. This system was demonstrated with surface plasmon resonance, impedance spectroscopy and chronoamperometry measurements, obtaining higher sensitivity (30 fM) with impedance spectroscopy.     

DNA sensors using a ferrocene-oligonucleotide conjugate

Using the redox-active DNA conjugate (ferrocene-modified oligonucleotide 12 mer) as a probe, the novel electrochemical gene sensor, which is sensitive, convenient, and not relying on radio isotope, has been developed. Oligonucleotide (16 mer) complementary to the target (19 mer) was immobilized onto gold electrode through the specific chemisorption of successive phosphorothioates which were introduced into 5'-end of the oligonucleotide. The sequence of the conjugate was designed to be also complementary to another site of the target. Therefore, the conjugate and the oligonucleotide anchoring on the electrode formed a sandwich-type ternary complex with a target DNA to give electric currents based on the ferrocene oxidation. By using this system, we have distinguished the mutant that has one base substitution from the fully complementary target.     

Adenosine Reagentless Electrochemical Aptasensor Using a Phosphorothioate Immobilization Strategy

This work deals with the characterization of a phosphorothioate anchoring strategy for aptamer molecules linked to gold, in the context of electrochemical sensors, using adenosine aptamer as model system. Surface density of immobilized phosphorothioate oligonucleotide sequences has been explored for a range of oligonucleotide concentrations (0.055–55 μM), finding a consequent variation of molecular surface density (3.5×10¹¹–2.8×10¹³ molecules/cm²). Most suitable aptamer concentration for adenosine recognition was also explored and found to be around 5.5 μM. As proof of concept of phosphorothioate strategy, electrochemical response to adenosine concentration was measured using a ferrocene‐labeled oligonucleotide sequence, and phosphorothioate anchoring thermal stability was compared to thiol immobilization.     

Probing of enzyme reactions by the biocatalyst-induced association or dissociation of redox labels linked to monolayer-functionalized electrodes

The activities of the enzymes tyrosinase and thrombin are probed by the association of the ferrocene boronic acid label to the enzyme-generated catechol ligand, and by the cleavage of the ligand-redox complex tethered to a peptide, respectively.     

Simple Methods for Preparing Oligonucleotides Containing 1) Thiol Groups at their 5′-Termini, 2) 5′-Phosphates

Abstract

A simple method has been developed for synthesising oligonucleotides containing a thiol group at their 5′-termini. The sequence required is prepared using standard solid phase phosphoramidite methods and an extra round of synthesis is then performed with S-triphenylmethyl O-methoxymorpholino-phosphinyl 3-mercaptopropan (1) ol. After normal deblocking this gives an oligonucleotide containing a tritylthiol group attached to the 5′-phosphate of an oligonucleotide via a 3-carbon spacer arm. The trityl group can be removed with AgNO₃ at pH 5 to give the free thiol. This compound is stable at pH 8 and reacts cleanly and rapidly with sulphydryl specific probes (eg fluorescent iodoacetates) at this pH value. This method can be used to prepare a wide variety of usefully labelled oligonucleotides and it is envisaged that fluorescent oligonucleotides will be useful in the study of protein nucleic acid interactions and to replace ³²P labelled hybridisation probes.     

Dot-blot amperometric genosensor for detecting a novel determinant of beta-lactamase resistance in Staphylococcus aureus

A new electrochemical hybridisation genosensor for the detection of resistant bacteria has been developed. This device relies on the immobilisation of a 50-mer oligonucleotide target, unique to a novel determinant of beta-lactamase resistance in Staphylococcus aureus, onto an electrochemical transducer. This genosensor is based on a concept adapted from classical dot-blot DNA analysis, but implemented in an electrochemical biosensor configuration. Amperometric transduction and an enzyme label method, that increases the genosensor sensitivity, are the main features of this new approach. In addition to the adapted dot-blot format, a double hybridisation assay, in which two different labelled probes were used, is reported. This procedure, if combined with polymerase chain reaction (PCR), allows determination of the genotype of an antibiotic-resistant organism in a shorter time than that required to perform traditional phenotypic susceptibility testing. Its characteristics are ideal for implementation in a kit form.     

Voltammetric Immunoassay for the Detection of Protein Biomarkers

A strategy for a fast (ca. 20 min), specific, electrochemical immunoassay for the cardiac biomarker creatine kinase (CK) and the human cytokine interleukin 10 (IL10) has been developed in this paper. The polyaniline modified gold surface formed from electrochemical reduction of diazonium salt supplies a solid substrate to link the activated carboxylic acid groups from the antibodies, which were labelled with ferrocene. The direct electrochemistry of ferrocene allows the analysis of protein markers with good sensitivity. The creatine kinase sensor demonstrates limit of detection of 0.5 pg mL^?1 in a physiological Krebs‐Henseleit solution. The anti‐IL10 antibody retained fluorescence activity after further coupling to ferrocene and covalent immobilization on to a gold electrode, showing a linear detection range for IL‐10 from 0.001 ng mL^?1 to 50 ng mL^?1 in PBS. We attribute the high sensitivity to the well‐controlled modified surface which results in end–on antibodies that can specifically capture the antigen with ease.     

Immunoaffinity for IgG antibodies of protein A labelled with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid

Summary Protein A was covalently labelled with 4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid in several molar ratios. The binding affinity for IgG and the delayed fluorescence of the corresponding complexes with Eu(III) were determined for each labelled compound. The immunoaffinity decreased slowly with increase in ratio of the label. A better immunoaffinity/labelling ratio was obtained by coupling protein A to the chelate via bovine serum albumin. A suitable ratio of chelating agent to protein A for producing a universal marker to be used in time-resolved fluoroimmunoassays can be determined.     

Aptamer-based electrochemical sensors that are not based on the target binding-induced conformational change of aptamers

This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors.     

Electrochemical Label‐Free Nucleotide Sensors

Numerous researchers have devoted a great deal of effort over the last few decades to the development of electrochemical oligonucleotide detection techniques, owing to their advantages of simple design, inherently small dimensions, and low power requirements. Their simplicity and rapidity of detection makes label‐free oligonucleotide sensors of great potential use as first‐aid screening tools in the analytical field of environmental measurements and healthcare management. This review article covers label‐free oligonucleotide sensors, focusing specifically on topical electrochemical techniques, including intrinsic redox reaction of bases, conductive polymers, the use of electrochemical indicators, and highly ordered probe structures.     

Experimental evidence for an isotropic distribution of cross-linkages in polystyrene model-networks

Polystyrene model-networks containing ferrocene labelled cross-linkages have been synthesized by anionic block copolymerization. A study by low-angle X-ray diffraction confirms the isotropic distribution of cross-linkages and the isotropic deformation of the network on swelling.     

Multiplexed detection of six labelled oligonucleotides using surface enhanced resonance Raman scattering (SERRS)

The labelling of target biomolecules followed by detection using some form of optical spectroscopy has become common practice to aid in their detection. This approach has allowed the field of bioanalysis to dramatically expand; however, most methods suffer from the lack of the ability to discriminate between the components of a complex mixture. Currently, fluorescence spectroscopy is the method of choice but its ability to multiplex is greatly hampered by the broad overlapping spectra which are obtained. Surface enhanced resonance Raman scattering (SERRS) holds many advantages over fluorescence both in sensitivity and, more importantly here, in its ability to identify components in a mixture without separation due to the sharp fingerprint spectra obtained. Here the first multiplexed simultaneous detection of six different DNA sequences, corresponding to different strains of the Escherichia coli bacterium, each labelled with a different commercially available dye label (ROX, HEX, FAM, TET, Cy3, or TAMRA) is reported. This was achieved with the aid of multivariate analysis, also known as chemometrics, which can involve the application of a wide range of statistical and data analysis methods. In this study, both exploratory discriminant analysis and supervised learning, by partial least squares (PLS) regression, were used and the ability to discriminate whether a particular labelled oligonucleotide was present or absent in a mixture was achieved using PLS with very high sensitivity (0.98-1), specificity (0.98-1), accuracy (range 0.99-1), and precision (0.98-1).