Analytical Application Of Aptamers Based On Capillary Electrophoresis: The Methodology Study And Preliminary Results In Diagnosis Of Primary Hepatic Carcinoma

Background and objectives: Nucleic acid aptamers are oligonucleotide ligandsbinding to targets with high affinity and specificity, which are screened from asynthetically random oligonucleotide library by systematic evolution of ligands byexponential enrichment (SELEX). We previously selected a group of aptamers withpooled liver cancer serum as target. Capillary electrophoresis (CE) is a microanalysistechnology with advantages of high efficiency, automation and rapidness. Thepresent study was aimed at establishing a CE-based method to analyze the bindingsituation of aptamers to serum and providing a new approach for the application ofaptamers in diagnosis of primary hepatic carcinoma.Methods:(1) Nucleic acid aptamers: Aptamers against liver cancer serum previouslyselected were synthesized and labeled with5’-FAM by a biological company.(2) Specimen collection: The serum samples were collected from the patientswith primary hepatic carcinoma and normal people and pooled liver cancer serumand pooled normal serum were prepared.(3) The reproducibility of aptamer analysis by CE: Reduplicate tubes ofaptamer solution were run by CE and the reproducibility of results was evaluated bycoefficient of variation. The analytical reproducibility of aptamers incubated withpooled liver cancer serum was also evaluated identically.(4) The factors influencing aptamer analysis by CE: A series of aptamerquantity was incubated with liver cancer serum and normal serum and run by CE tooptimize aptamer amount used. The optimal amount of aptamers was incubated withserum in different temperatures and run by CE to optimize incubation temperature.The optimal amount of aptamers was incubated with serum in the optimaltemperature and run by CE in different voltage to optimize voltage. The optimalamount of aptamers was incubated with serum at optimal temperature and sampledwith various times before running by CE at optimal voltage to optimize samplingtime. Finally, under the above optimal conditions, CE analysis was performed withdifferent running buffer solutions to determine the suitable buffer solutions.(5) Orthogonal experiment for optimal condition of CE analysis: On the basisof above optimization,4factors influencing results obviously,4levels each, wereselected to design the orthogonal experiment for optimizing the condition of aptmaer analysis be CE. Aptamers were incubated with pooled liver cancer serum and poolednormal serum, respectively, and run be CE according to the orthogonal table. Eachtest was repeated three times.(6) Diagnostic value of aptamer analysis by CE: Serum specimens wereanalyzed with the aptamers by CE under optimal conditions and its diagnosis valueon primary hepatic carcinoma was assessed. The peak aera and the area ratio ofpeaks were analyzed by receiver operating characteristic (ROC) curve, by which thearea under the curve (AUC) was obtained and the cut-off value was determined tocalculate sensitivity, specificity and accuracy for aptamers to diagnose liver cancer.Results:(1) The reproducibility of aptamer analysis by CE: The coefficients ofvariation were4.48%in CE analysis of10reduplicate tests of aptamer solution and4.93%in CE analysis of10reduplicate tests of aptamers incubated with pooled livercancer serum.(2) The factors influencing aptamer analysis by CE: The optimal amount ofaptamer was0.9pmol, the appropriate incubation temperature was25℃, the optimalisolation voltage was25KV, the appropriate sampling time was5s, and the suitablebuffer was phosphate buffer solution.(3) The results of orthogonal experiment: The areas of peak C and peak B ofliver cancer serum was larger than that of normal serum. With the area ratio of peakC and peak B as indicators for statistical analysis, we have determined the optimalconditions for analysis of aptamers by CE, that is, the pH of buffer was8, the voltagewas15kV, the incubation temperature was25℃and aptamer amount is0.5pmol.(4) The value of aptamer for the diagnosis of hepatic carcinoma: We havedetected111cases of liver cancer serum and61cases of normal serum under aboveoptimized conditions. The differences of the area of peak B and the area ration ofpeak C and peak B between liver cancer group and normal group were significant(P=0.000). These two parameters were valuable in diagnosis of liver cancer withAUC0.930and0.925, sensitivity94.6%and92.0%, specificity82.0%and85.2%,and accuracy82.5%and82.7%, respectively.Conclusions:In this study, we observed the factors influencing the binding of aptamer toserum in CE analysis and optimized the conditions for aptamer analysis by CE withorthogonal experiment. A CE-based method to determine the binding situation of aptamers to serum was successfully established, which is valuable in the diagnosis ofprimary hepatic carcinoma.