Electrochemical DNA biosensors based on platinum nanoparticles combined carbon nanotubes

Platinum nanoparticles were used in combination with multi-walled carbon nanotubes (MWCNTs) for fabricating sensitivity-enhanced electrochemical DNA biosensor. Multi-walled carbon nanotubes and platinum nanoparticles were dispersed in Nafion, which were used to fabricate the modification of the glassy carbon electrode (GCE) surface. Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated daunomycin. Due to the ability of carbon nanotubes to promote electron-transfer reactions, the high catalytic activities of platinum nanoparticles for chemical reactions, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.0 × 10⁻¹¹ mol l⁻¹.     

DNA hybridization assays using temperature gradient focusing and peptide nucleic acids

Two types of DNA hybridization assays are demonstrated with temperature gradient focusing (TGF) and peptide nucleic acids (PNAs). In TGF, the application of a controlled temperature gradient along the length of a microchannel filled with an appropriate temperature-dependent buffer results in the formation of a gradient in both the electric field and electrophoretic velocity. Ionic species move in this gradient and concentrate at a unique point where the total velocity sums to zero. The first assay is a mixing assay in which PNA is allowed to flow through spatially focused DNA targets within a capillary. The second assay detects single base pair mutations (SBPM) by monitoring the fluorescence intensity of PNA/DNA duplexes as a function of temperature within the capillary. The SBPM analysis can be performed in less than 5 min with 100-fold more dilute analyte compared to conventional UV melting measurements.     

Gold nanoparticle based signal enhancement liquid crystal biosensors for DNA hybridization assays

A novel signal enhanced liquid crystal biosensor based on using AuNPs for highly sensitive DNA detection has been developed. This biosensor not only significantly decreases the detection limit, but also offers a simple detection process and shows a good selectivity to distinguish perfectly matched target DNA from two-base mismatched DNA.     

Electrochemical and piezoelectric DNA biosensors for hybridisation detection

DNA biosensors (or genosensors) are analytical devices that result from the integration of a sequence-specific probe and a signal transducer. Among other techniques, electrochemical and piezoelectric methods have recently emerged as the most attractive due to their simplicity, low instrumentation costs, possibility for real-time and label-free detection and generally high sensitivity.Focusing on the most recent activity of worldwide researchers, the aim of the present review is to give the readers a critical overview of some important aspects that contribute in creating successful genosensing devices. Advantages and disadvantages of different sensing materials, probe immobilisation chemistries, hybridisation conditions, transducing principles and amplification strategies will be discussed in detail. Dedicated sections will also address the issues of probe design and real samples pre-treatment. Special emphasis will be finally given to those protocols that, being implemented into an array format, are already penetrating the molecular diagnostics market.     

The interaction of poly(ethylenimine) with nucleic acids and its use in determination of nucleic acids based on light scattering

For the first time, poly(ethylenimine) (PEI) was used to determine nucleic acids with a light scattering technique using a common spectrofluorometer. The interaction of PEI with DNA results in greatly enhanced intensity of light scattering at 300 nm, which is caused by the formation of the big particles between DNA and PEI. Based on this, a new quantitative method for nucleic acid determination in aqueous solutions has been developed. Under the optimum conditions, the enhanced intensity of light scattering is proportional to the concentration of nucleic acid in the range of 0.01-10.0 microg ml(-1) for herring sperm DNA (hsDNA), 0.02-10.0 microg ml(-1) for calf thymus DNA (ctDNA), 0.02-20.0 microg ml(-1) for yeast RNA (yRNA). The detection limits are 5.3, 9.9, and 13.7 ng ml(-1), respectively. Synthetic samples were determined satisfactorily. At the same time, the light scattering technique has been successfully used to obtain the information on the effects of pH and ionic strength on the formation and the stability of the DNA/PEI complex, which is important in some fields such as genetic engineering and gene transfer. Using ethidium bromide (EB) as a fluorescent probe, the binding of PEI with hsDNA was studied. Both the binding constant of EB with DNA and the number of binding sites per nucleotide decrease with increasing concentration of PEI, indicating noncompetitive inhibition of EB binding to DNA in the presence of PEI. And the association constant of PEI to DNA obtained is 1.2 x 10(5) M(-1). IR-spectra show that PEI interacts with DNA through both the phosphate groups and the bases of DNA and the formation of DNA/PEI complex may cause the change of the conformation of the DNA secondary structure, which is also proved by UV-spectra.     

Cationic modified nucleic acids for use in DNA hairpins and parallel triplexes

Non-nucleosidic DNA monomers comprising partially protonated amines at low pH have been designed and synthesized. The modifications were incorporated into DNA oligonucleotides via standard DNA phosphoramidite synthesis. The ability of cationic modifications to stabilize palindromic DNA hairpins and parallel triplexes were evaluated using gel electrophoresis, circular dichroism and thermal denaturation measurements. The non-nucleosidic modifications were found to increase the thermal stability of palindromic hairpins at pH 8.0 as compared with a nucleosidic tetraloop (TCTC). Incorporation of modifications at the 5'-end of a triplex forming oligonucleotide resulted in a significant increase in thermal stability at low pH when the modifications were placed as the 5'-dangling end.     

Application of peptide nucleic acids containing azobenzene self-assembled electrochemical biosensors in detecting DNA sequences

Hybridization of peptide nucleic acids probe containing azobenzene (NH₂-TNT₄, N-PNAs) with DNA was performed by covalently immobilizing of NH₂-TNT₄ in sequence on the 3-mercaptopropionic acid self-assembled monolayer modified gold electrode with the helps of N-(3-dimethylaminopropy1)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the hybrid was coded as N-PNAs/DNA. Using [Fe(CN)₆]^4−/3− (1:1) as the electrochemical indicator, the electrochemical properties of the N-PNAs self-assembled monolayer (N-PNAs-SAMs) and N-PNAs/DNA hybridization system under the conditions of before and after UV light irradiation were characterized with cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectra (EIS). Results showed that the redox currents decreased with the increase of irradiation time, suggesting that the ability of the charge transfer on the electrode surface was weakened and the conformation of hybrid system had been changed, and the control of PNAs/DNA hybridization could be realized by UV light irradiation. Supported by the National Natural Science Foundation of China (Grant No. 50572107) and “Top Hundred Talents Program” of Chinese Academy of Science     

Electrochemical biosensors in nonaqueous solutions and their applications.

This review first describes the invention of functional interfaces to promote biochemical redox reactions between substrates in dipolar aprotic solvents and enzymes or related compounds immobilized at the interface. The interfaces contain hydrophilic polymer membranes, a gold nanoparticle self-assembled electrode constructed by using rigid rod dithiols, and binary self-assembled monolayers composed of amino and carboxyl terminal groups. Other topics covered are: the electrochemical characterization of the hydrophilic polymer membrane; the development of biosensors to obtain reaction parameters of enzymatic and of electrochemical kinetics; and applications to the study of materials involved in metabolism.     

Recombinant antibodies and their use in biosensors

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.     

Electrochemical biosensors based on enzymes immobilized in electropolymerized films

A review based on 135 references concerns the design and properties of electrochemical biosensors for 13 different substrates of enzymatic reactions. In the sensors discussed the enzymes are immobilized within or on the top of electropolymerized films, mostly of conducting polymers. Amperometric detection is most often used for internal electrochemical sensing.Dedicated to our late colleague Wojciech Matuszewski     

Electrochemical sensors and biosensors based on heterogeneous carbon materials

Abstract  

An overview of the use of electrochemical sensors made from heterogeneous carbon materials (carbon paste electrodes, screen-printed carbon electrodes) in the field of food analysis is presented. Sensors for inorganic and organic analytes as well as biosensors are summarized.     

Electrochemical sensors and biosensors based on heterogeneous carbon materials

Abstract  An overview of the use of electrochemical sensors made from heterogeneous carbon materials (carbon paste electrodes, screen-printed carbon electrodes) in the field of food analysis is presented. Sensors for inorganic and organic analytes as well as biosensors are summarized. Graphical abstract        

Electrochemical biosensor for detection of BCR/ABL fusion gene based on hairpin locked nucleic acids probe

This communication reports on a novel biosensor to study the hybridization specificity by using thiolated hairpin locked nucleic acids (LNA) as the capture probe. The LNA probe was immobilized on the gold electrode through sulfur–Au interaction and could selectively hybridize with its target DNA. Differential pulse voltammetry (DPV) was used to monitor the hybridization reaction on the probe electrode. The decrease of the peak current of methylene blue, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This LNA probe has been used for assay of fusion gene in Chronic Myelogenous Leukemia (CML) of the real sample with satisfactory result.     

DNA biosensors based on metallo-intercalator probes and electrocatalytic amplification

Strategies for electrochemical sensing of DNA can be classified into label-free and label-based approaches, categories of which include enzyme-, nanomaterial- and redox labels that are attached to DNA either by covalent or non-covalent means. Metallointercalators represent one group of small molecule redox labels that non-covalently enter the groove of a DNA. The metallointercalator plays a dual-role in acting as a structure indicator (for hybridization) and a signal generator. Labeling is not needed, and electrochemical measurements can be carried out in a label-free solution of an electrolyte. However, such metallointercalators lack the option of catalytic signal generation as in the case of enzyme- and nanomaterial-based labels. Therefore, signal amplification becomes crucial. We first survey here recent progress in this area. A signal-amplifying system is presented that relies on the electroatalytic oxidation of a metallointercalator ruthenium(II)bipyridine/phenoxazine complex in the presence of electron donor species such as oxalate, DNA bases, or tripropylamine. Recent work on such DNA sensors is discussed. Results suggest that such metallointercalator-based DNA sensors represent a viable platform for developing high-throughput and automated PCR/lab-on-a-chip devices as well as visualized multifunctional DNA sensors.

Duplex probes: a new approach for the detection of specific nucleic acids in homogenous assays

A novel method for the detection of specific nucleic acids in homogenous solution was developed. The method is based on the use of duplex probes in which fluorescent donor and quencher labeled on either oligonucleotide are held in close proximity, so that fluorescence is quenched. Amplification of the target sequence results in the cleavage of the probe and the resulting fluorescence can be detected. The fluorescent data analysis demonstrated that the duplex probes can specifically recognize the presence of target, and a significantly higher lever of relative fluorescent signal than TaqMan probes is obtainable. Combined with real-time PCR instruments, the assay can be used to quantify the input target molecules. As few as five copies of initial target molecules can be detected, and a large dynamic linear ranger (five orders of magnitude) is obtained.     

Electrochemical biosensors for detection of point mutation based on surface ligation reaction and oligonucleotides modified gold nanoparticles

An electrochemical method for point mutation detection based on surface ligation reaction and oligonucleotides (ODNs) modified gold nanoparticles (AuNPs) was demonstrated. Point mutation identification was achieved using Escherichia coli DNA ligase. This system for point mutation detection relied on a sandwich assay comprising capture ODN immobilized on Au electrodes, target ODN and ligation ODN. Because of the sequence-specific surface reactions of E. coli DNA ligase, the ligation ODN covalently linked to the capture ODN only in the presence of a perfectly complementary target ODN. The presence of ligation products on Au electrode was detected using chronocoulometry through hybridization with reporter ODN modified AuNPs. The use of AuNPs improved the sensitivity of chronocoulometry in this approach, a detection limit of 0.9 pM complementary ODN was obtained. For single base mismatched ODN (smODN), a negligible signal was observed. Even if the concentration ratio of complementary ODN to smODN was decreased to 1:1000, a detectable signal was observed. This work may provide a specific, sensitive and cost-efficient approach for point mutant detection.     

Molecular orbital calculations on the conformation of nucleic acids and their constituents

The conformational properties of the furanose ring of purine- and pyrimidine--nucleosides and-nucleotides are studied quantum-mechanically with the help of the PCILO method, using the pseudorotational concept. The computations point to the existence of two stable conformational zones centered around the C(3)-endo and C(2)-endo conformations which in the isolated furanose ring are separated by barriers of the order of 4 kcal/mole. In nucleosides one of the barriers (the one running through the O(1)-exo-C(2)-exo path) becomes very high. A detailed study is made of the relation between the phase angle of pseudorotation, P, and the torsion angle about the glycosyl bond, _CN. A very satisfactory agreement with the available experimental data is observed.

---

Zusammenfassung Die Konformationseigenschaften des Furanoserings in -Nucleosiden und Nucleotiden von Purin und Pyrimidin werden nach der PCILO-Methode unter Berücksichtigung der pseudorotatorischen Betrachtungsweise studiert. Die Rechnung läßt auf die Existenz zweier stabiler Konformationszonen schließen, die in der Umgebung der C(2)-endo und der C(3)-endo Konformationen liegen, und die im isolierten Furanosering durch Energiebarrieren der Größenordnung von 4 kcal/mol voneinander getrennt sind. In Nucleosiden wird eine der Barrieren (die durch den Weg O(1)-exo-C(2)-exo gekennzeichnete) sehr hoch. Die Relation zwischen dem Phasenwinkel der Pseudorotation, P, und dem Drehwinkel um die Glycosylbindung, _CN, wird einer eingehenden Untersuchung unterworfen. Man beobachtet eine sehr zufriedenstellende Übereinstimmung mit den verfügbaren experimentellen Daten.

---

Résumé Les propriétés conformationnelles du noyau furanose des -nucleosides et nucleotides des purines et pyrimidines sont étudiées par la méthode PCILO en faisant appel au concept de la pseudorotation. Les calculs indiquent l'existence de deux zones de conformations stables, centrées autour des conformations C(2)-endo et C(3)-endo, qui sont dans le sucre isolé séparées par des barrières de l'ordre de 4 kcal/mole. Dans les nucleosides, l'une de ces barrières (celle qui passe par le chemin O(1)-exo-C(2)-exo) devient très élevée. Une étude détaillée est effectuée sur la relation entre l'angle de phase de la pseudorotation P et l'angle de torsion autour de la liaison glycosylique, _CN. Un excellent accord avec les données expérimentales disponibles est observé.

---

---

This research was supported by the R.C.P. 173 and the A.T.P. A 655-2303 of the C.N.R.S.     

Electrochemical biosensors based on Ti3C2Tx MXene: future perspectives for on-site analysis

MXenes are recently developed two-dimensional layered materials composed of early transition metal carbides and/or nitrides that provide unique characteristics for biosensor applications. This review presents the recent progress made on the usage and applications of MXenes in the field of electrochemical biosensors, including microfluidic biosensors and wearable microfluidic biosensors, and highlights the challenges with possible solutions and future needs. The multilayered configuration and high conductivity make these materials as an immobilization matrix for the biomolecule immobilization with activity retention and to be explored in the fabrication of electrochemical sensors, respectively. First, how the MXene nanocomposite as an electrode modifier affects the sensing performance of the electrochemical biosensors based on enzymes, aptamer/DNA, and immunoassays is well described. Second, recent developments in MXene nanocomposites as wearable biosensing platforms for the biomolecule detection are highlighted. This review pointed out the future concerns and directions for the use of MXene nanocomposites to fabricate advanced electrochemical biosensors with high sensitivity and selectivity. Specifically, possibilities for developing microfluidic electrochemical sensors and wearable electrochemical microfluidic sensors with integrated biomolecule detection are emphasized.     

DNA circuits as amplifiers for the detection of nucleic acids on a paperfluidic platform

This article describes the use of non-enzymatic nucleic acid circuits based on strand exchange reactions to detect target sequences on a paperfluidic platform. The DNA circuits that were implemented include a non-enzymatic amplifier and transduction to a fluorescent reporter; these yield an order of magnitude improvement in detection of an input nucleic acid signal. To further improve signal amplification and detection, we integrated the enzyme-free amplifier with loop-mediated isothermal amplification (LAMP). By bridging the gap between the low concentrations of LAMP amplicons and the limits of fluorescence detection, the non-enzymatic amplifier allowed us to detect as few as 1200 input templates in a paperfluidic format.