Abstract
► Simultaneous detection of two functional genes in the same system was studied. ► PCR and restriction endonuclease digestion enhanced the sensitivity of sensor. ► Horseradish peroxidase and laccase labels were used for signal amplification. ► Application for simultaneous detection of MnP and CDH genes from white rot fungi. ► The results of DNA detection were consistent with UV spectrometry.
An electrochemical DNA sensor for simultaneous detection of functional genes encoding manganese peroxidase (MnP) and cellobiose dehydrogenase (CDH) on a gold electrode was developed. After two thiolated capture probes assembled on the electrode surface, the electrode was exposed to a monolayer of 6-mercapto-1-hexanol (MCH) solution to prevent nonspecific adsorption of target DNA and detection probes. Horseradish peroxidase–streptavidin (HRP–SA) conjugate and laccase–streptavidin (LAC–SA) conjugate were applied for enzyme-amplified amperometric measurement. The two target genes were simultaneously quantified in the same system. The DNA conformation and surface coverage on electrode were characterized by impedance spectroscopy and cyclic voltammetry. The amperometric current responses to HRP and LAC-catalyzed reactions were linearly related to the common logarithm of two target nucleic acids concentrations, ranging from 1
×
10
−11
M to 4
×
10
−8
M and 1
×
10
−10
M to 4
×
10
−8
M. The correlation coefficients were 0.9884 and 0.9881, and the detection limits were 6.2
×
10
−12
M and 3.0
×
10
−11
M, respectively. The effectiveness of this DNA sensor was confirmed by simultaneous detection of two gene fragments extracted from
Phanerochaete chrysosporium using polymerase chain reaction (PCR) and restriction endonuclease digestion. The DNA biosensor exhibited good selectivity, precision, stability and reproducibility.