Development of aptamer-based methods for determination of carcinoembrionic antigen

In this study, aptamer-based methods were developed with two different techniques for the determination of carcinoembryonic antigen (CEA) protein, which is one of the cancer diagnostic techniques. Two different CEA-specific aptamers were used for each analytical technique. The first method is based on the determination of CEA by quartz crystal microbalance (QCM) technique after two separate selective aptamers are bonded to the piezocrystal with electrochemical nanofilm. The other method is CEA determination with the help of selective aptamers using the surface plasmon resonance enhanced total internal reflection ellipsometry (SPRe-TIRE) method. In this study, the CEA-specific aptamers were 5′- GGGG CGAC GTTG AGAT TCCG CTTG TGTA TTAG TAGT CCCC-3′ (antiCEA1, KD= 37.8 nM) and 5′-ATAC CAGC TTAT TCAA TTGG GGTA GGGG GCGA AGCG ATAC CCTA ATCA GC-3′ (antiCEA2, KD= 3.9 nM), respectively. After both aptamer platforms were characterized by various techniques, CEA analysis methods were developed for two different aptamers. In the CEA determination method developed with antiCEA1 and antiCEA2 aptamers using the QCM technique, the equations y=0.25452x+0.31227 and y=0.15113x+0.2659 were obtained, respectively, in the concentration range of 0.1-25 ng/mL. The detection limits of the methods (LOD, S/N=3) were determined as 0.102 ng/mL for the antiCEA1 platform and 1.416 ng/mL for the antiCEA2 platform. In the methods developed with the SPRe-TIRE technique, linear equations y=0.9764x+4.7766 and y=0.6571x+1.9496 were obtained, respectively, for antiCEA1 and antiCEA2 aptamer platforms in the concentration range of 0.01-500 ng/mL. The LOD values of the methods were determined as 0.1 and 2.9 pg/mL, respectively. In the regression analyzes performed in all methods were developed with both techniques, the R2 values ranged between 0.9870 and 0.9955. For antiCEA1 aptasensor platforms with higher analysis performance, the interference effects of α-fetoprotein (AFP), cancer antigen-125 (CEA-125) and vascular endothelial growth factor-165 (VEGF-165) species were investigated. Potentially interfering species were added at a concentration of 500 ng/mL in the presence of CEA, and the signal change due to interference was obtained for QCM and SPRe-TIRE in the range of 1.0% to 4.1%. In the validation tests of the developed antiCEA1 aptasensors, the standard addition method to human blood serum was used to represent real samples. The recovery values of the methods were found between 95.5% and 103.5% by spiked 0.1 and 10 ng/mL CEA for QCM technique and 1.00 and 50 ng/mL CEA for SPRe-TIRE technique.