RESUMEN
A novel electrochemical aptasensor was developed for ultrasensitive detection of staphylococcal enterotoxin B (SEB) by combining signal amplification and target-induced aptamer release strategy. A gold electrode was modified with a nanocomposite made of gold nanoparticles reduced in situ, zirconia nanoparticles, and chitosan. The SEB aptamer was hybridized by anchoring the capture probe on the modified gold electrode surface through AuS binding. In the presence of SEB, the capture probe-aptamer duplex was compelled to open, releasing the aptamer from the electrode. The resulting single-strand capture probe was hybridized with a biotinylated detection probe and labeled with streptavidin-horseradish peroxidase, producing an ultrasensitive enzyme-catalyzed electrochemical signal. Under optimal conditions, the amperometric responses were proportional to the SEB concentrations ranging from 2 to 512ngmL(-1), with a detection limit of as low as 0.24ngmL(-1) (S/N=3). The aptasensor exhibited good stability, outstanding reproducibility, and high selectivity. The as-prepared aptasensor was used to analyze SEB in human serum specimens, and validated through enzyme-linked immunosorbent assay method. Analytical results suggest that the developed assay is a promising alternative approach for detecting SEB in clinical diagnosis.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Quitosano/química , Técnicas Electroquímicas/métodos , Enterotoxinas/análisis , Oro/química , Nanopartículas del Metal/química , Circonio/química , Espectroscopía Dieléctrica , Electrodos , Ensayo de Inmunoadsorción Enzimática , Humanos , Nanopartículas del Metal/ultraestructura , Reproducibilidad de los ResultadosRESUMEN
A sensitive and selective electrochemical DNA sensor was developed for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML). Firstly, graphene sheets (GS) suspension was prepared with the aid of chitosan (CS) solution and then fabricated onto the glassy carbon electrode (GCE), followed by the electro-polymerization of aniline to form the PANI layer, then, Au nanoparticles (AuNPs) were electro-deposited onto the modified GCE to immobilize the capture probes. The capture probe employed a hairpin structure and dually labeled with a 5'-SH and a 3'-biotin. After hybridization with the target DNA, hairpin structure was compelled to open and 3'-biotin was forced to stay away from the electrode surface. As a result, streptavidin-alkaline phosphatase (SA-AP) was covalently binded to the capture probe via biotin-avidin system. Reduction currents were then generated after catalyzing the hydrolysis of the electroinactive 1-naphthyl phosphate (1-NP) to 1-naphthol and monitored by differential pulse voltammetry (DPV). Under optimum conditions, the amperometric signals increased linearly with the target DNA concentrations (10 pM to 1000 pM), and the DNA sensor exhibited a detection limit as low as 2.11 pM (S/N=3) with an excellent differentiation ability, and the proposed method showed acceptable stability and reproducibility. It has been applied for assay of BCR/ABL fusion gene from real samples with satisfactory results.
Asunto(s)
Compuestos de Anilina/química , ADN/genética , Técnicas Electroquímicas/métodos , Proteínas de Fusión bcr-abl/genética , Oro/química , Grafito/química , Nanopartículas/química , Técnicas Biosensibles/métodos , Línea Celular , ADN/análisis , Humanos , Nanopartículas/ultraestructura , Hibridación de Ácido Nucleico/métodos , Reproducibilidad de los ResultadosRESUMEN
A novel strategy to simplify the dehydrogenase-based electrochemical biosensor fabrication through one-step drop-coating nanobiocomposite on a screen printed electrode (SPE) was developed. The nanobiocomposite was prepared by successively adding graphitized mesoporous carbons (GMCs), meldola's blue (MDB), alcohol dehydrogenase (ADH) and cofactor nicotinamide adenine dinucleotide (NAD(+)) in chitosan (CS) solution. MDB/GMCs/CS film was prepared. Cyclic voltammetry measurements demonstrated that MDB was strongly adsorbed on GMCs. After optimizing the concentration of MDB and the working potential, the MDB/GMCs/CS film presented a fast amperometric response (5s), excellent sensitivity (10.36 nA µM(-1)), wide linear range (10-410 µM) toward NADH and without any other interference signals (such as AA, UA, DA, H2O2 and metal ions). Furthermore, concentrations of ADH and NAD(+) in nanobiocomposite and the detection conditions (temperature and pH) were also optimized. The constructed disposable ethanol biosensor showed an excellent linear response ranged from 0.5 to 15 mM with high sensitivity (67.28 nA mM(-1)) and a low limit of detection (80 µM) and a remarkable long-term stability (40 days). The intra-batch and inter-batch variation coefficients were both less than 5% (n=5). The ethanol recovery test demonstrated that the proposed biosensor offered a remarkable and accurate method for ethanol detection in the real blood samples.
Asunto(s)
Acetaldehído/metabolismo , Alcohol Deshidrogenasa/metabolismo , Técnicas Biosensibles/métodos , NAD/metabolismo , Nanocompuestos/química , Acetaldehído/sangre , Acetaldehído/química , Alcohol Deshidrogenasa/química , Técnicas Biosensibles/instrumentación , Carbono/química , Catálisis , Quitosano/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Grafito/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , NAD/química , Oxazinas/química , Reproducibilidad de los Resultados , Temperatura , Factores de TiempoRESUMEN
A novel electrochemical immunosensor for detecting toxoplasma gondii-specific IgM (Tg-IgM) was constructed based on goldmag (Au-Fe(3)O(4)) nanoparticles and graphene sheets (GS). Thionine (Thi), as a mediator, was first electropolymerized on a nafion-GS (Nf-GS) modified electrode. Subsequently, gold nanoparticles (AuNPs) were attached onto the poly-thionine film through π-stacking interactions, and then were used to immobilize toxoplasma gondii antigen (Tg-Ag) for immunosensor fabrication. A sandwich-type immunoassay for Tg-IgM was performed using Au-Fe(3)O(4) labeled anti-IgM-horseradish peroxidase (HRP) as trace label. Electrochemical detection was carried out in the presence of H(2)O(2) as HRP substrate. Using Au-Fe(3)O(4) provided a simple, non-chemical damaging method for regeneration, and enhanced the HRP reduction ability toward H(2)O(2). The AuNPs/Thi/Nf-GS nanocomposite also had good conductivity and biocompatibility, which effectively improved the immunosensor sensitivity. Under optimal conditions, the immunosensor can detect Tg-IgM in two linear ranges from 0.0375 to 1.2 AU mL(-1) and from 2.0 to 18 AU mL(-1) with a detection limit of 0.016 AU mL(-1) (S/N=3). The immunosensor exhibited good reproducibility, stability, and selectivity as well.