RESUMO
A nanowell array electrode-based electrochemical quantitative system without amplification was developed and applied for the detection of H5N1 target DNA. An 18-mer probe was immobilized on a nanowell array electrode with a diameter of 500 nm, which was coated with streptavidin and a self-assembly monolayer (SAM). The surface properties of probe DNA hybridization with complementary target DNA were characterized using atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The AFM image shows that the depth of nanowell was reduced from 200 nm to 15 nm due to the formation of a DNA hybridization complex on the streptavidin/SAM structure. Differences in charge transfer resistance (deltaR(ct)) in EIS upon hybridization of the probe DNA with complementary target DNA were analyzed and used for the quantitation of H5N1 DNA. This approach shows that the quantitative analysis of H5N1 DNA ranging from 1 pM to 1 microM DNA is possible on a nanowell array electrode.
Assuntos
DNA Viral/química , Virus da Influenza A Subtipo H5N1 , Nanotecnologia/métodos , Hibridização de Ácido Nucleico , Biotinilação , Impedância Elétrica , Eletroquímica , Eletrodos , Desenho de Equipamento , Teste de Materiais , Microscopia de Força Atômica , Propriedades de SuperfícieRESUMO
The rapid identification and verification of single nucleotide polymorphisms (SNPs) were demonstrated using a well array sensor containing anti-biofouling titanium (Ti). Probe single-stranded DNA (ssDNA) was immobilized inside a titanium-well array on amine-modified glass surfaces with anti-biofouling behavior via a streptavidin-biotin interaction. Fluorescence intensity changes originating from the hybridization of nucleic acids to protein-bound nucleic acids linked to Alexa Fluor (FL) 647 were observed. The protocol was highly sensitive and reproducible for the detection of DNA hybridization. Significant changes in fluorescence signals were observed when using target DNA with a single base mismatch, indicating that this method is applicable to SNP detection. The microarray technology for the detection of SNPs using anti-biofouling Ti and other methods can be used as a highly sensitive in vitro medical sensor, as highlighted by an increase in genotyping accuracy.
Assuntos
Análise Mutacional de DNA/instrumentação , DNA/genética , Nanotecnologia/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA/instrumentação , Titânio/química , Desenho de Equipamento , Análise de Falha de Equipamento , Espectrometria de Fluorescência/instrumentaçãoRESUMO
An electrochemical microfluidic device has been fabricated on PET (polyethylene terephthalate) substrate using an imprinting method. The imprinting transfers patterns from a stamp into a substrate mechanically. However, a blanket mould imprinting process has been introduced to embed the photolithographically produced gold metal electrode lines into the PET substrate resulting in an individually addressable array flush to better than 100 nm. The device formed one wall of a packed chromatography column. The array was electrochemically characterised using standard redox probes in both stagnant conditions and under flow. Both numerical modelling and experimental data show improved sensitivity under flow and a limiting current which scaled linearly with the cube root of the volume flow rate. A chromatographic separation of the bioanalytical significant neurotransmitter dopamine (DA) and its metabolite DOPAC was achieved and electrochemically detected at multiple locations within the column. The PET device was stable and robust to leaks to pressures well in excess of those required for chromatographic separations.
