Double-tagging polymerase chain reaction with a thiolated primer and electrochemical genosensing based on gold nanocomposite sensor for food safety.
A novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. This assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomole range. Rigid conducting gold nanocomposite represents a good material for the improved and oriented immobilization of biomolecules with excellent transducing properties for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors.
Marques
Paulo R Brasil de Oliveira
PR
UNESP, Universidade Estadual Paulista, Instituto de Quimica, Laboratorio de Eletroanalitica, Campus de Araraquara, São Paulo, Brazil.
Lermo
Anabel
A
Campoy
Susana
S
Yamanaka
Hideko
H
Barbé
Jordi
J
Alegret
Salvador
S
Pividori
M Isabel
MI
D000818:Animals; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004076:Digoxigenin / Q000737:chemistry / Q000378:metabolism; D004563:Electrochemistry; D004566:Electrodes; D005504:Food Analysis / Q000379:methods*; D005516:Food Microbiology; D016680:Genome, Bacterial; D006046:Gold / Q000737:chemistry*; D053761:Nanocomposites / Q000737:chemistry*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013499:Surface Properties; D013997:Time Factors
D017931:DNA Primers; D004269:DNA, Bacterial; D013438:Sulfhydryl Compounds; D006046:Gold; D004076:Digoxigenin