Ultra-sensitive detection of mutated papillary thyroid carcinoma DNA using square wave stripping voltammetry method and amplified gold nanoparticle biomarkers.
This study presents an ultra-sensitive technique for the electrochemical detection of the mutated BRAF gene associated with papillary thyroid carcinomas (PTC). In the proposed approach, a biotinylated 30-nucleotides probe DNA was immobilized in a streptavidin-modified 96-well microtiter plate and the free active sites of the streptavidin were blocked using biotinylated bovine serum albumin (BSA). The biotinylated target DNA was then added and allowed to hybridize with the immobilized probe DNA for 30min. Subsequently, streptavidin-labeled gold nanoparticles were added, and a nanoparticle enlargement process was performed using gold ion solution and formaldehyde reductant. The gold particles were then dissolved in bromide and DNA hybridization detection process was performed using a square wave stripping voltammetry (SWSV) technique. The results indicated a stable SWSV response in differential detection between blank solution and target DNA solution with a concentration of 130aM. Moreover, the coefficient of determination (R(2)) of the semi-log plot of the SWSV response current against the target DNA concentration (0.52-1300aM) was found to be 0.9982. The detection limit was estimated to be 0.35aM (based on a signal-to-noise ratio of 3:1). This value was approximately three orders of magnitude lower than that obtained using the same method but without gold amplification process. Finally, the proposed approach is successful in differentiating between the mutant and wildtype BRAF sequences that are present in genuine 224-nucleotides DNA.
Liao
Kuo-Tang
KT
Department of Chemistry, National Sun Yat-Sen University, 804, Kaohsiung, Taiwan, ROC.
Cheng
Jiin-Tsuey
JT
Li
Chun-Liang
CL
Liu
Rue-Tsuan
RT
Huang
Hsuan-Jung
HJ
D000231:Adenocarcinoma, Papillary / Q000235:genetics*; D015374:Biosensing Techniques / Q000295:instrumentation*; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry*; D048493:Proto-Oncogene Proteins B-raf / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013964:Thyroid Neoplasms / Q000235:genetics*
D004273:DNA, Neoplasm; D006046:Gold; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf