RESUMEN
A simple strategy for covalent immobilizing DNA sequences, based on the formation of stable diazonized conducting platforms, is described. The electrochemical reduction of 4-nitrobenzenediazonium salt onto screen-printed carbon electrodes (SPCE) in aqueous media gives rise to terminal grafted amino groups. The presence of primary aromatic amines allows the formation of diazonium cations capable to react with the amines present at the DNA capture probe. As a comparison a second strategy based on the binding of aminated DNA capture probes to the developed diazonized conducting platforms through a crosslinking agent was also employed. The resulting DNA sensing platforms were characterized by cyclic voltammetry, electrochemical impedance spectroscopy and spectroscopic ellipsometry. The hybridization event with the complementary sequence was detected using hexaamineruthenium (III) chloride as electrochemical indicator. Finally, they were applied to the analysis of a 145-bp sequence from the human gene MRP3, reaching a detection limit of 210 pg µL(-1).
Asunto(s)
ADN/análisis , Compuestos de Diazonio/química , Hibridación de Ácido Nucleico/métodos , Técnicas Biosensibles , Carbono/química , Sondas de ADN/química , Técnicas Electroquímicas , Electrodos , Humanos , Límite de DetecciónRESUMEN
An electrochemical DNA genosensor constructed by using rough gold as electrode support is reported in this work. The electrode surface nanopatterning was accomplished by repetitive square-wave perturbing potential (RSWPP). A synthetic 25-mer DNA capture probe, modified at the 5' end with a hexaalkylthiol, able to hybridize with a specific sequence of lacZ gene from the Enterobacteriaceae bacterial family was assembled to the rough gold surface. A 25 bases synthetic sequence fully complementary to the thiolated DNA capture probe and a 326 bases fragment of lacZ containing a fully matched sequence with the capture probe, which was amplified by a specific asymmetric polymerase chain reaction (aPCR), were employed as target sequences. The hybridization event was electrochemically monitored by using two different indicators, hexaammineruthenium (III) chloride showing an electrostatic DNA binding mode, and pentaamineruthenium-[3-(2-phenanthren-9-yl-vinyl)-pyridine] (in brief RuL) which binds to double stranded DNA (dsDNA) following an intercalative mechanism. After optimization of the different variables involved in the hybridization and detection reactions, detection limits of 5.30 pg µL(-1) and 10 pg µL(-1) were obtained for the 25-mer synthetic target DNA and the aPCR amplicon, respectively. A RSD value of 6% was obtained for measurements carried out with 3 different genosensors prepared in the same manner.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas Electroquímicas/instrumentación , Electrodos , Nanoestructuras , Técnicas Biosensibles/métodos , Complejos de Coordinación/química , Técnicas Electroquímicas/métodos , Enterobacteriaceae/genética , Oro , Operón Lac , Hibridación de Ácido Nucleico , Oligonucleótidos/análisis , Reacción en Cadena de la Polimerasa , Compuestos de Rutenio , Sensibilidad y Especificidad , Electricidad EstáticaRESUMEN
We report the in situ generation of aryl diazonium cations of Azure A, a redox-active phenothiazine dye, by reaction between the corresponding aromatic aminophenyl group and sodium nitrite in 0.1 M HCl. The subsequent electrochemical reduction of these dye diazonium salts gives rise to conductive electrografted films onto screen-printed carbon (SPC) electrodes. The resulting Azure A films have a very stable and reversible electrochemical response and exhibit potent and persistent electrocatalytic behavior toward NADH oxidation. We have optimized the electrografting conditions in order to obtain SPC modified electrodes with high and stable electrocatalytic response. The kinetic of the reaction between the NADH and the redox active centers in the Azure A film has been characterized using cyclic voltammetry and single step chronoamperometry. The catalytic currents were proportional to the concentration of NADH giving rise to linear calibration plots up to a concentration of 0.5 mM with a detection limit of 0.57±0.03 µM and a sensitivity of 9.48 A mol cm(-2) µM(-1). The precision of chronoamperometric determinations was found to be 2.3% for five replicate determinations of 3.95 µM NADH. The great stability of such modified electrodes makes them ideal for their application in the development of biosensing platforms based on dehydrogenases.
Asunto(s)
Colorantes Azulados/química , Compuestos de Diazonio/síntesis química , Técnicas Electroquímicas/instrumentación , NAD/análisis , Carbono/química , Técnicas Electroquímicas/métodos , Electrodos , Cinética , Oxidación-Reducción , Oxidorreductasas , Reproducibilidad de los Resultados , Nitrito de Sodio/químicaRESUMEN
A new electrochemical approach for an accurate quantification of DNA base pairs in genomic human DNA amplified by polymerase chain reaction (PCR) is described. The method is based on the immobilization of the sample (a thiolated DNA fragment) on the surface of a screen-printed gold electrode through the -SH group at the 5'-end and the subsequent intercalation of a ruthenium pentaamin complex as a redox indicator. The determination of the base pair number in the sequence is achieved by measuring the changes in the electroactivity of the ruthenium complex using Differential Pulse Voltammetry. Calibration curves correlating current intensity with the base pair number allow determining the size of DNA samples, even when very large (over 100 base pairs) sequences are assayed. The method has been successfully applied to detect the DNA cleavage by a site-specific restriction enzyme. The electrochemical approach developed offers the advantage of ease of performance in comparison to other previously described approaches, which are time-consuming and require sophisticated and expensive instrumentation.
Asunto(s)
Emparejamiento Base , División del ADN , ADN/química , Desoxirribonucleasa HpaII/química , Técnicas Electroquímicas , Electrodos , Oro , Humanos , Compuestos de Rutenio/químicaRESUMEN
A screening method for rapid detection of gene mutations directly in polymerase chain reaction (PCR) of genomic DNA is described. The method involves the development of a disposable screen-printed gold electrode modified with a thiolated capture probe directly obtained from denaturated PCR genomic DNA, which recognizes (by hybridization) its fully complementary sequence (wild type), giving a signal, whereas no signal is obtained for single-mismatched target (mutant). The detection of the hybridization event is achieved by changes in the metal redox center electroactivity of the complex [Ru(NH(3))(5) L](2+), where L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], at -0.200 V. This complex binds to double-stranded DNA in a very selective form. The method allows discrimination between the wild type and the mutant of gene MRP3 directly in large PCR amplicons extracted from blood cells, without the need to use either synthetic probes or labeled targets. The mutation involves the presence of a single-nucleotide polymorphism (SNP) at base 54 of a 145-base-pair sequence from exon 21 of gene MRP3. Since the presence of this SNP might lead to a variety of hereditary liver disorders, its identification in a rapid and easy form may provide novel therapeutic targets for the future. The screening method proposed has excellent signal reproducibility, with a relative standard deviation of 10%. In addition, with the method developed as little as 6.6 ng/muL PCR product can be detected.
Asunto(s)
Técnicas Biosensibles , Mutación , Secuencia de Bases , ADN/genética , Sondas de ADN , Electroquímica , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido SimpleRESUMEN
Selective polynucleotide recognition and detection based on a dual-stage method are described. The method involves the development of a recognition surface based on gold nanoparticles modified with a thiolated capture probe able to hybridize with its complementary sequence (target). After hybridization, this sensing surface is removed from the solution and electrodeposited on an electrode surface. The detection of the hybridization event is achieved using the complex [Ru(NH(3))(5)L](2+), were L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], as electrochemical indicator. This complex binds to double strand DNA more efficiently than to single stranded DNA. The advantage of this dual-stage DNA sensing method is the high selectivity derived from the separation of the hybridization event (occurring on one surface) from the detection step (on a different surface), enabling the analysis of long target DNAs, which is usually the case in real DNA sequence analysis. In addition, this approach not only quantifies pmol of a complementary target sequence but also is sensitive to the presence of a single mismatch and its position in the sequence.
Asunto(s)
Técnicas Biosensibles , ADN/análisis , ADN/genética , Oro/química , Nanopartículas , Compuestos Organometálicos/química , Técnicas Electroquímicas , Electrodos , Hibridación de Ácido NucleicoRESUMEN
The preparation of DNA-sensing architectures based on gold nanoparticles (Au-NPs) in conjunction with an "in situ" prepared ruthenium complex as a new route to improve the analytical properties of genosensors is described. In the development of these architectures several strategies to obtain Au-NPs modified gold electrodes (Au-NP/Au) have been essayed, in particular covalent binding and electrochemical deposition from a solution containing Au-NPs previously synthesized. UV-vis absorption measurements in conjunction with transmission electron microscope (TEM) images reveal that the synthesized Au-NPs are stable for at least 4 weeks and have a narrow size distribution. Atomic force microscopy (AFM) was employed to characterize the morphology and to estimate the Au-NPs surface coverage of the modified gold electrodes obtained following the different modification strategies. In order to assess the utility of these architectures as DNA-sensing devices, a thiolated capture probe sequence from Helicobacter pylori was immobilized onto the as-prepared surface. This sequence was chosen as a case of study within the framework of developing approaches of wide applicability. The hybridization event is detected using a water-soluble pentaamin ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] complex (Ru(NH(3))(5)L) prepared "in situ". This complex, due to its intercalative character, is able to bind to double stranded DNA more efficiently than to single stranded DNA. In addition, the metal provides with a redox center that can be used as an electrochemical indicator. On the basis of this strategy, complementary target sequences of H. pylori have been detected over the range of 40-800 pmol with a detection limit of 25+/-2 pmol.
Asunto(s)
Técnicas Biosensibles/instrumentación , ADN Bacteriano/genética , Oro/química , Helicobacter pylori/genética , Nanopartículas/química , Rutenio/química , Análisis de Secuencia de ADN/instrumentación , Mapeo Cromosómico/instrumentación , ADN Bacteriano/análisis , Diseño de Equipo , Análisis de Falla de Equipo , Nanopartículas/ultraestructura , Nanotecnología/instrumentación , Tamaño de la Partícula , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A ruthenium complex, pentaamine ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] (which we refer to as RuL in the text) generated in situ has been used as a sensitive and selective electrochemical indicator in DNA sensing. The complex incorporates dual functionalities with the Ru center providing a redox probe and the ligand (L) providing a fluorescent tag. The presence of the aromatic groups in the ligand endows the complex with an intercalative character and makes it capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Combining spectroscopic and electrochemical techniques, we have elucidated the nature of the interactions. From these data we conclude that the binding mode is fundamentally intercalative. The ligand-based fluorescence allows characterization of the complex formation as well as for melting experiments to be carried out. The metal-based redox center is employed as an electrochemical indicator to detect the hybridization event in a DNA biosensor. The biosensor has been developed by immobilization of a thiolated capture probe sequence from Helicobacter pylori onto gold electrodes. With the use of this approach, complementary target sequences of Helicobacter can be quantified over the range of 106 to 708 pmol with a detection limit of 92+/-0.4 pmol and a linear correlation coefficient of 0.995. In addition, this approach allows the detection, without the need for a hybridization suppressor in solution, such as formamide, of not only a single mismatch but also its position in a specific sequence of H. pylori, due to the selective interaction of this bifunctional ruthenium complex with dsDNA.