RESUMEN
Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe. Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.
Asunto(s)
Bacterias/clasificación , Bacterias/aislamiento & purificación , Técnicas Bacteriológicas/instrumentación , Técnicas Electroquímicas/instrumentación , Bacterias/genética , Técnicas Bacteriológicas/métodos , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , ARN Ribosómico/análisis , ARN Ribosómico/genéticaRESUMEN
Precursor rRNA (pre-rRNA) is an intermediate stage in the formation of mature rRNA and is a useful marker for cellular metabolism and growth rate. We developed an electrochemical sensor assay for Escherichia coli pre-rRNA involving hybridization of capture and detector probes with tail sections that are spliced away during rRNA maturation. A ternary self-assembled monolayer (SAM) prepared on gold electrode surfaces by coassembly of thiolated capture probes with hexanedithiol and posttreatment with 6-mercapto-1-hexanol minimized the background signal and maximized the signal-to-noise ratio. Inclusion of internal calibration controls allowed accurate estimation of the pre-rRNA copy number per cell. As expected, the ratio of pre-rRNA to mature rRNA was low during stationary phase and high during log phase. Pre-rRNA levels were highly dynamic, ranging from 2 copies per cell during stationary phase to ~1,200 copies per cell within 60 min of inoculation into fresh growth medium. Specificity of the assay for pre-rRNA was validated using rifampin and chloramphenicol, which are known inhibitors of pre-rRNA synthesis and processing, respectively. The DNA gyrase inhibitor, ciprofloxacin, was found to act similarly to rifampin; a decline in pre-rRNA was detectable within 15 min in ciprofloxacin-susceptible bacteria. Assays for pre-rRNA provide insight into cellular metabolism and are promising predictors of antibiotic susceptibility.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Escherichia coli/genética , Precursores del ARN/análisis , ARN Ribosómico/análisis , Antiinfecciosos/metabolismo , Cloranfenicol/metabolismo , Ciprofloxacina/metabolismo , Farmacorresistencia Bacteriana/genética , Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Precursores del ARN/biosíntesis , Precursores del ARN/metabolismo , ARN Ribosómico/biosíntesis , Rifampin/metabolismo , Inhibidores de Topoisomerasa IIRESUMEN
We report the concentration and purification of bacterial 16S rRNA by the use of a biotinylated DNA target-specific capture (TSC) probe. For both cultivated bacterial and urine specimens from urinary tract infection patients, TSC resulted in a 5- to 8-fold improvement in the sensitivity of bacterial detection in a 16S rRNA electrochemical sensor assay.
Asunto(s)
Bacterias/clasificación , Bacterias/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Sondas de Oligonucleótidos , ARN Ribosómico 16S/aislamiento & purificación , Bacterias/genética , Humanos , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Orina/microbiologíaRESUMEN
A ternary surface monolayer, consisting of coassembled thiolated capture probe, mercaptohexanol and dithiothreitol, is shown to offer dramatic improvements in the signal-to-noise characteristics of electrochemical DNA hybridization biosensors based on common self-assembled monolayers. Remarkably low detection limits down to 40 zmol (in 4 µL samples) as well as only 1 CFU Escherichia coli per sensor are thus obtained without any additional amplification step in connection to the commonly used horseradish peroxidase/3,3',5,5'-tetramethylbenzidine system. Such dramatic improvements in the detection limits (compared to those of common binary alkanethiol interfaces and to those of most electrochemical DNA sensing strategies without target or signal amplification) are attributed primarily to the remarkably higher resistance to nonspecific adsorption. This reflects the highly compact layer (with lower pinhole density) produced by the coupling of the cyclic- and linear-configuration "backfillers" that leads to a remarkably low background noise even in the presence of complex sample matrixes. A wide range of surface compositions have been investigated, and the ternary mixed monolayer has been systematically optimized. Detailed impedance spectroscopy and cyclic voltammetric studies shed useful insights into the surface coverage. The impressive sensitivity and high specificity of the simple developed methodology indicate great promise for a wide range of nucleic acid testing, including clinical diagnostics, biothreat detection, food safety, and forensic analysis.
Asunto(s)
Técnicas Biosensibles/métodos , ADN/análisis , Ditioeritritol/química , Hibridación de Ácido Nucleico/métodos , Compuestos de Sulfhidrilo/química , Bencidinas/química , Técnicas Electroquímicas/métodos , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/diagnóstico , Humanos , Límite de Detección , Masculino , ARN Bacteriano/análisisRESUMEN
Here, we report that the smooth muscle and endothelium of the pig coronary artery differ in the profiles of energy metabolism nucleotides. ATP levels in the freshly isolated smooth muscle (1490 +/- 93, all the values are in pmol/mg protein) were significantly greater than in the endothelium (418 +/- 68). In contrast, endothelium contained higher levels of NADH (328 +/- 21), NAD+ (1210 -/+ 28), NADPH (87 +/- 2), and NADP+ (77 +/- 4) than smooth muscle (17 +/- 2, 96 +/- 14, 7 +/- 1, and 8 +/- 1, respectively). However, smooth muscle and endothelium do not differ from each other in the ratios of NADH/NAD+ or NADPH/NADP+. Cells cultured from smooth muscle and endothelium contained less ATP (93 +/- 2, 141 +/- 6) and had lower ratios of NADH/ NAD+ than the freshly isolated tissues but the NADPH/NADP+ ratios remained similar. We conclude that (a) freshly isolated smooth muscle and endothelium differ in their profiles of the energy metabolism nucleotides, and (b) culturing the cells alters the profile.