RESUMEN
Thrombin, a serine protease playing a central role in the coagulation cascade reactions and a potent neurotoxin produced by injured brain endothelial cells, is a recognized cardiac biomarker and a critical biomarker for Alzheimer's disease development. Both in vivo and in vitro, its low physiological concentrations and nonspecific binding of other components of physiological fluids complicate electroanalysis of thrombin. Here, femtomolar levels of thrombin in serum and an artificial cerebrospinal fluid (CSF) were detected by the indicator-free electrochemical methodology exploiting the O2 reduction reaction directly, with no electron transfer mediators, electrocatalyzed by the covalent G4-hemin DNAzyme complex naturally self-assembling upon thrombin binding to the hemin-modified 29-mer DNA aptamer sequence tethered to gold via an alkanethiol linker. Coadsorbed PEG inhibited nonspecific protein binding and allowed the sought signal resolution. The proposed assay exploiting the "oxidase" activity of G4-hemin DNAzyme does not require any coreactants necessary for the traditional peroxidase activity-based assays with this DNAzyme, such as H2O2 and redox mediators, or solution deaeration and allows fast, overall 30 min analysis of thrombin in aerated buffer, CSF, and 1% human serum solutions. This pioneer approach exploiting the oxidase activity G4-hemin DNAzyme is simple, sensitive, and selective and represents a new tool for ultrasensitive electrocatalytic assays based on simple and efficient O2-dependent DNAzyme labels.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Trombina/análisis , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/metabolismo , Biomarcadores/análisis , ADN Catalítico/química , Hemina/química , Humanos , MasculinoRESUMEN
Improved outcomes for many types of cancer achieved during recent years is due, among other factors, to the earlier detection of tumours and the greater availability of screening tests. With this, non-invasive, fast and accurate diagnostic devices for cancer diagnosis strongly improve the quality of healthcare by delivering screening results in the most cost-effective and safe way. Biosensors for cancer diagnostics exploiting aptamers offer several important advantages over traditional antibodies-based assays, such as the in-vitro aptamer production, their inexpensive and easy chemical synthesis and modification, and excellent thermal stability. On the other hand, electrochemical biosensing approaches allow sensitive, accurate and inexpensive way of sensing, due to the rapid detection with lower costs, smaller equipment size and lower power requirements. This review presents an up-to-date assessment of the recent design strategies and analytical performance of the electrochemical aptamer-based biosensors for cancer diagnosis and their future perspectives in cancer diagnostics.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Neoplasias , Anticuerpos , Técnicas Electroquímicas , Humanos , Neoplasias/diagnósticoRESUMEN
In this report, an electrochemical immunosensor for the selective and sensitive monitoring of Aß1-42 fibrils is presented. The sensing platform was prepared by the formation of a 4,4'-thiobisbenzenethiol (TBBT) self-assembled monolayer on a clean gold surface followed by the covalent entrapment of gold nanoparticles (AuNPs). The half-antibody fragments of the Anti-Amyloid Fibrils antibody were immobilized on AuNPs via S-Au covalent bonds. Each step of immunosensor fabrication was characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The biosensor was successfully used for the sensing of Aß1-42 fibrils in both phosphate saline buffer (PBS) and artificial blood plasma (ABP). The immunosensor sensitivity estimated based on calibration slopes was better in the presence of APP in the comparison to PBS. The LOD values obtained for both measuring media were of 0.6 pM level. The moderate response towards Aß1-42 oligomers demonstrated the immunosensor selectivity.
Asunto(s)
Péptidos beta-Amiloides/sangre , Sustitutos Sanguíneos/química , Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Péptidos/sangre , Anticuerpos Inmovilizados , Calibración , Espectroscopía Dieléctrica , Técnicas Electroquímicas/instrumentación , Electrodos , Oro/química , Límite de Detección , Nanopartículas del Metal/química , Plasma/químicaRESUMEN
We applied a cobalt-porphyrin modified DNA as electrochemical marker, which was attached to nanoparticles, to detect specific DNA sequences. We compare the performance of gold and silver NPs in oligonucleotide sensors to determine if a change in metal will lead to either higher sensitivity or different selectivity, based on the redox behaviour of silver vs. gold. Surprisingly, we find that using either gold or silver NPs yields very similar overall performance. The electrochemical measurements of both types of sensors show the same redox behaviour which is dominated by the cobalt porphyrin, indicating that the electron pathway does not include the NP, but there is direct electron transfer between the porphyrin and the electrode. Both sensors show a linear response in the range of 5 × 10-17-1 × 10-16 M; the limit of detection (LOD) is 3.8 × 10-18 M for the AuNP sensor, and 5.0 × 10-18 M for the AgNP sensor, respectively, which corresponds to the detection of about 20-50 DNA molecules in the analyte. Overall, the silver system results in a better DNA economy and using cheaper starting materials for the NPs, thus shows better cost-effectivness and could be more suitable for the mass-production of highly sensitive DNA sensors.
Asunto(s)
Técnicas Biosensibles/métodos , ADN/química , ADN/genética , Oro/química , Nanopartículas del Metal/química , Metaloporfirinas/química , Plata/química , Secuencia de Bases , Electroquímica , Límite de Detección , Modelos LinealesRESUMEN
The background: The monolayers self-assembled on the gold electrode incorporated transition metal complexes can act both as receptor ("host" molecules) immobilization sites, as well as transducer for interface recognitions of "guest" molecules present in the aqueous solutions. Their electrochemical parameters influencing the sensing properties strongly depend on the transition metal complex structures. The objectives: The electrochemical characterization of the symmetric terpyridine-M2+-terpyridine and asymmetric dipyrromethene-M2+-terpyridine complexes modified with ssDNA probe covalently attached to the gold electrodes and exploring their ssDNA sensing ability were the main aims of the research presented. The methods: Two transition metal cations have been selected: Cu2+ and Co2+ for creation of redox-active monolayers. The electron transfer coefficients indicating the reversibility and electron transfer rate constant measuring kinetic of redox reactions have been determined for all SAMs studied using: Cyclic Voltammetry, Osteryoung Square-Wave Voltammetry, and Differential Pulse Voltammetry. All redox-active platforms have been applied for immobilization of ssDNA probe. Next, their sensing properties towards complementary DNA target have been explored electrochemically. The results: All SAMs studied were stable displaying quasi-reversible redox activity. The linear relationships between cathodic and anodic current vs. san rate were obtained for both symmetric and asymmetric SAMs incorporating Co2+ and Cu2+, indicating that oxidized and reduced redox sites are adsorbed on the electrode surface. The ssDNA sensing ability were observed in the fM concentration range. The low responses towards non-complementary ssDNA sequences provided evidences for sensors good selectivity. The conclusions: All redox-active SAMs modified with a ssDNA probe were suitable for sensing of ssDNA target, with very good sensitivity in fM range and very good selectivity. The detection limits obtained for SAMs incorporating Cu2+, both symmetric and asymmetric, were better in comparison to SAMs incorporating Co2+. Thus, selection of the right transition metal cation has stronger influence on ssDNA sensing ability, than complex structures.
Asunto(s)
Técnicas Biosensibles/métodos , ADN de Cadena Simple/análisis , Electrodos , Oro/química , ADN de Cadena Simple/química , Técnicas Electroquímicas , Humanos , Cinética , Límite de Detección , Oxidación-ReducciónRESUMEN
An electrochemical genosensor based on an epoxy-phenanthroline-Fe(III)-NH2-ssDNA layer for the detection of RNA derived from Avian Influenza is presented. The biosensor preparation consists of: (I) modification of gold electrodes with aminoethanethiol, (II) modification of the self-assembled monolayer of aminoethanethiol with 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline using "click" chemistry, (III) a first step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (IV) a second step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (V) immobilization of the single stranded amino-DNA probe via "click" chemistry between epoxy and amino groups. The interactions between the ssDNA probe and RNA targets were explored with Osteryoung Square Wave Voltammetry. The genosensor showed a remarkable detection limit of 3 copies/µL (5 aM) for RNA extracted from A/swan/Poland/305/06 (H5N1) containing a fully complementary sequence. A linear dynamic range for this sequence was observed from 3.0×103 to 3.0×105 [copies/µl]. RNA extracted from A/mallard/Poland/446/09 (H7N7), containing a non-complementary sequence, generated a much weaker response. Moreover, the developed genosensor allows to distinguish RNA present in biological samples having 2, 3 and 4 mismatches. This biosensing approach can become a potential alternative tool for detecting RNA samples in biomedical research and early clinical diagnosis of avian influenza viruses.
Asunto(s)
Secuencia de Bases , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Virus de la Influenza A/genética , Gripe Aviar/virología , Análisis de Secuencia de ARN/métodos , Animales , Técnicas Biosensibles/instrumentación , Embrión de Pollo , ADN de Cadena Simple/química , Técnicas Electroquímicas/instrumentación , Electrodos , Compuestos Epoxi/química , Compuestos Férricos , Oro , Virus de la Influenza A/aislamiento & purificación , Polonia , Aves de Corral/virología , ARN Complementario , ARN Viral/química , Sensibilidad y EspecificidadRESUMEN
In cancer diagnostics, specific analysis of blood-circulating proteins biomarkers of cancer is often complicated both by their inherently low concentrations and by strong interference from serum/blood proteins. Here, we report a simple and robust electrochemical cellulase-linked sandwich assay on magnetic beads (MBs) for fM-sensitive analysis of the Human Epidermal growth factor Receptor-2 HER-2/neu protein that is over-expressed in most aggressive breast cancers. In the assay, a sandwich is assembled by capturing HER-2/neu on either antibody (Ab) or aptamer-modified MBs accompanied by reaction with the second Ab or aptamer labelled with cellulase. On application of the sandwiches assembled on MBs onto a cost-effective graphite electrode modified with an insulating nitrocellulose film, the cellulase label digests the film. This results in the pronounced changes in the electrical properties of the modified electrodes. The chronocoulometrically-measured extent of the produced changes was proportional to the 10-15-10-10â¯M HER-2/neu in the analyzed samples, and down to 1â¯fM of HER-2/neu could be detected in human serum samples in an overall less than 3â¯h assay. The developed simple and electrochemically label-free methodology is general and can be easily adapted for testing of any other protein.
Asunto(s)
Biomarcadores de Tumor/sangre , Técnicas Electroquímicas/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Receptor ErbB-2/sangre , Aptámeros de Nucleótidos/química , Secuencia de Bases , Celulasa/química , ADN/química , Técnicas Electroquímicas/instrumentación , Electrodos , Grafito/química , Humanos , Fenómenos MagnéticosRESUMEN
We describe an ultrasensitive electrochemical genosensor based on gold nanoparticles and cobalt-porphyrin functionalised ssDNA probes. The sensitivity at the attomolar concentration level arises from an increased density of redox labels on the electrode surface compared to sensors without NP modification. The electrode detects as few as 23 DNA molecules, approaching single molecule detection.
Asunto(s)
Técnicas Biosensibles/métodos , ADN de Cadena Simple/análisis , ADN de Cadena Simple/química , Oro/química , Nanopartículas del Metal/química , Metaloporfirinas/química , Adsorción , Cobalto/química , ADN de Cadena Simple/genética , Técnicas Electroquímicas/métodos , Electrodos , Límite de Detección , Hibridación de Ácido Nucleico , Oxidación-Reducción , Tamaño de la PartículaRESUMEN
This paper concerns the development of a redox-active monolayer and its application for the construction of an electrochemical genosensor designed for the detection of specific DNA and RNA oligonucleotide sequences related to the avian influenza virus (AIV) type H5N1. This new redox layer was created on a gold electrode surface step by step. Cyclic Voltammetry, Osteryoung Square-Wave Voltammetry and Differential Pulse Voltammetry were used for its characterization. This new redox-active layer was applied for the construction of the DNA biosensor. The NH2-NC3 probe (20-mer) was covalently attached to the gold electrode surface via a "click" reaction between the amine and an epoxide group. The hybridization process was monitored using the Osteryoung Square-Wave Voltammetry. The 20-mer DNA and ca. 280-mer RNA oligonucleotides were used as the targets. The constructed genosensor was capable to determine complementary oligonucleotide sequences with a detection limit in the pM range. It is able to distinguish the different position of the part RNA complementary to the DNA probe. The genosensor was very selective. The 20-mer DNA as well as the 280-mer RNA oligonucleotides without a complementary sequence generated a weak signal.
Asunto(s)
Aves/virología , Técnicas Electroquímicas/métodos , Subtipo H5N1 del Virus de la Influenza A/genética , Gripe Aviar/diagnóstico , ARN Viral/análisis , Aminación , Animales , Técnicas Biosensibles , Sondas de ADN/química , Sondas de ADN/genética , Técnicas Electroquímicas/instrumentación , Electrodos , Compuestos Epoxi/química , Oro/química , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Hibridación de Ácido Nucleico/métodos , Oxidación-Reducción , Fenantrolinas/química , ARN Viral/genéticaRESUMEN
A DNA biosensor for detection of specific oligonucleotides sequences of Plum Pox Virus (PPV) in plant extracts and buffer is proposed. The working principles of a genosensor are based on the ion-channel mechanism. The NH2-ssDNA probe was deposited onto a glassy carbon electrode surface to form an amide bond between the carboxyl group of oxidized electrode surface and amino group from ssDNA probe. The analytical signals generated as a result of hybridization were registered in Osteryoung square wave voltammetry in the presence of [Fe(CN)6]3-/4- as a redox marker. The 22-mer and 42-mer complementary ssDNA sequences derived from PPV and DNA samples from plants infected with PPV were used as targets. Similar detection limits of 2.4 pM (31.0 pg/mL) and 2.3 pM (29.5 pg/mL) in the concentration range 1-8 pM were observed in the presence of the 22-mer ssDNA and 42-mer complementary ssDNA sequences of PPV, respectively. The genosensor was capable of discriminating between samples consisting of extracts from healthy plants and leaf extracts from infected plants in the concentration range 10-50 pg/mL. The detection limit was 12.8 pg/mL. The genosensor displayed good selectivity and sensitivity. The 20-mer partially complementary DNA sequences with four complementary bases and DNA samples from healthy plants used as negative controls generated low signal.
Asunto(s)
Técnicas Biosensibles , ADN Viral/aislamiento & purificación , Virus Eruptivo de la Ciruela/aislamiento & purificación , Secuencia de Bases/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/aislamiento & purificación , ADN Viral/genética , Límite de Detección , Hibridación de Ácido Nucleico , Extractos Vegetales/química , Extractos Vegetales/genética , Virus Eruptivo de la Ciruela/genéticaRESUMEN
Electrochemical biosensors have emerged as reliable analytical devices suitable for pathogen detection. Low cost, small sample requirement and possibility of miniaturization justifies their increasing development. Thus, we report in this review on the state of the art of avian influenza virus detection with genosensors and immunosensors working by an electrochemical mode. Their working principles focusing on the physical properties of the transducer, the immobilization chemistry, as well as new trends including incorporation of nanoparticles will be presented. Then, we critically review the detection of avian influenza virus in the complex matrices that use electrochemical biosensors and compare them with traditionally applied methods such as ELISA or Western blot.
Asunto(s)
Técnicas Biosensibles , Electroquímica/métodos , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/diagnóstico , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Humanos , Gripe Humana/virología , Nanopartículas , Sensibilidad y EspecificidadRESUMEN
The duo-genosensor consisting of two different oligonucleotide probes immobilized covalently on the surface of one gold electrode via Au-S bond formation was used for simultaneous determination of two different oligonucleotide targets. One of the probes, decorated on its 5'-end with ferrocene (SH-ssDNA-Fc), is complementary to the cDNA representing a sequence encoding part of H5 hemagglutinin from H5N1 virus. The second probe, decorated on its 5'-end with methylene blue (SH-ssDNA-MB), is complementary to cDNA representing the fragment of N1 neuraminidase from the same virus. The presence of both probes on the surface of gold electrodes was confirmed with Osteryoung square-wave voltammetry (OSWV). The changes in redox activity of both redox active complexes before and after the hybridization process were used as analytical signal. The peak at +400 ± 2 mV was observed in the presence of 40 nM ssDNA used as a target for SH-ssDNA-Fc probe. This peak increased with the increase of concentration of target ssDNA. It indicates the "signal on" mode of analytical signal generation. The peak at -250 ± 4 mV, characteristic for SH-ssDNA-MB probe, was decreasing with the increase of the concentration of the complementary ssDNA target starting from 8 to 100 nM. This indicates the generation of electrochemical signal according to the "signal off" mode. The proposed duo-genosensor is capable of simultaneous, specific, and good sensitivity probing for the sequences derived from genes encoding two main markers of the influenza virus, hemagglutinin and neuraminidase.