RESUMO
A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7 A/M) and rapid (45 min) detection of low nanomolar levels of synthetic target DNA (25 fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.
Assuntos
Técnicas Eletroquímicas/métodos , Escherichia coli/genética , Ácidos Nucleicos Imobilizados/química , Hibridização de Ácido Nucleico/métodos , RNA Bacteriano/análise , RNA Ribossômico/análise , Dióxido de Silício/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , DNA/análise , DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Desenho de Equipamento , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Humanos , Ácidos Nucleicos Imobilizados/genética , Porosidade , RNA Bacteriano/genética , RNA Ribossômico/genéticaRESUMO
In this work, we combined a hierarchical nano-array effect of silicon nanowires (SiNWs) with a metallic surface of silver nanoparticles (AgNPs) to design a surface-enhanced Raman spectroscopy (SERS) scattering substrate for sensitive detection of Rhodamine 6G (R6G) which is a typical dye for fluorescence probes. The SiNWs were prepared by Metal-Assisted Chemical Etching (MACE) of n-Si (100) wafers. The Doehlert design methodology was used for planning the experiment and analyzing the experimental results. Thanks to this methodology, the R6G SERS response has been optimized by studying the effects of the silver nitrate concentration, silver nitrate and R6G immersion times and their interactions. The immersion time in R6G solution stands out as the most of influential factor on the SERS response.
RESUMO
The demand for on-site nanodevices is constantly increasing. The technology development for the design of such devices is highly regarded. In this work, we report the design of a disposable platform that is structured with cauliflower-shaped gold nanoparticles (cfAuNPs) and we show its applications in immunosensing and enzyme-based detection. The electrochemical reduction of Au(III) allows for the electrodeposition of highly dispersed cauliflower-shaped gold nanoparticles on the surface of screen-printed carbon electrodes (SPCEs). The nanostructures were functionalized using ferrocenylmethyl lipoic acid ester which allowed for the tethering of the ferrocene group to gold, which serves as an electrochemical transducer/mediator. The bioconjugation of the surface with anti-human IgG antibody (α-hIgG) or horseradish peroxidase (HRP) enzyme yields biosensors, which have been applied for the selective electrochemical detection of human IgG (hIgG) or H2O2 as model analytes, respectively. Parameters such as the number of sweeps, amount of charge generated from the oxidation of the electrodeposited gold, time of incubation and concentration of the ferrocene derivatives have been studied using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Selectivity and specificity tests have been also performed in the presence of potentially interfering substances to either hIgG or H2O2. Results showed that the devised immunosensor is endowed with good selectivity and specificity in the presence of several folds of competitive analytes. The enzyme-based platform showed a good catalytic activity towards H2O2 oxidation which predestined it to potential applications pertaining to enzymatic kinetics studies. The levels of hIgG in human serum and H2O2 in honey were successfully determined and served as assessment tools of the applicability of the platforms for real samples analysis.