Detection Of Carbohydrate Antigen 15-3 By Optical Protein-Chip Based On Imaging Ellipsometry Biosensor

OBJECTIVEBased on imaging ellipsometry and multi-microarray protein-chip technique (automatic analysis system), the optical protein-chip is a new technique for protein detection with advantages of label-free, high through-put, etc.. In this study, we have tried to use this technique for the detection of serum carbohrydrate antigen 15-3 (CA 15-3), a clinical routine used tumor marker, and measured CA 15-3 in serum samples. The results were compared with those by Electrochemiluminescence method. Besides, the clinical significance of CA 15-3 concentrations in different groups was discussed.METHODSSubject: 80 human serum samples (in which CA15-3 was tested by electrochemiluminescence) were randomly drawn out from the serum samples afforded by Affiliated Hospital of Shandong Academy of Medical Science. There were 18 controls (with no tumors) aged 51±3 years old and 62 cancer patients aged 60±11 years oldThe device of ellipsometry imaging system, optical protein-chip system (which including devices of protein microarray, micro-pump system), software for ellipsometric visualization and analysis, etc., were used in present study. Experimental materials used mainly were: silicon wafers, surface modification reagents, antigen and antibodies of CA 15-3 (from Shanghai Linc-Bio Science Co.LTD and Sigma). Silicon wafers were cut, washed, surface modified by carboxylic reagents, and then they were used for preparing the CA 15-3 antigen detection protein-chips. After selection of the proper antibody of CA 15-3, we have tested the method for detecting CA 15-3 antigen by protein-chips and setup its detective standar curve of concentration (CA 15-3 antigen)–gray (ellipsometric visualization counts). The accuracy and sensitivity were tested as well. Then with the function of relationship between concentration and gray counts, we tested CA 15-3 antigen by our optical protein chip.Statistical analysis softwares of SPSS 18.0, Stata/SE 11.0 for windows, Wilicoxon rank sum Test, Receiver Operating Characteristic Curve (ROC) and paired t-test were used for analysis the data and comparisons of data between groups (control and cancer) and methods (optical protein-chip and electrochemiluminescence). Significancyα=0.05.RESULTSBased on the results of antibodies selection, CA 15-3 antibody from Shanghai Linc-Bio Science Co.LTD was chosen for preparing the CA 15-3 detective optical protein chip. The method of detecting CA 15-3 by optical protein chip was established and its accuracy and sensitivity were tested. The lowest threshold for detecting CA 15-3 was 5.30 IU/mL and the detection showed good repeatability, as its relative standard deviation values were 7.67%, 10.57% and 9.40% at 46.33 IU/mL, 23.16 IU/mL and 11.58 IU/mL, respectively (n=6).We had setup a standard curve of concentration-gray for detecting CA 15-3 by optical protein chip, its function was calculated as, y = 1.23x+ 87.616,and Spearman's correlation coefficient (r) was 0.97, and the detecting limitation of this curve was 5.80 IU/mL, which could meet the clinical demands. The concentrations of CA 15-3 in 80 serum samples were detected by our optical protein chips. The average concentrations of CA 15-3 were 2.84±0.73 IU/ml and 52.45±7.50 IU/ml in controls and cancer patients groups, respectively, and the difference was statistically significant(P<0.05).The Areas under ROC curves of detecting serum CA 15-3 concentration by electrochemiluminescence and optical protein chip showed no significant difference between the two methods(P>0.05). The comparison by paired t-test (P=0.78) showed the same result as well. Therefore, detecting of CA 15-3 was in accordance by the two methods.CONCLUSIONS1. We have established primarily a new method for detecting CA 15-3 by optical protein-chip based on imaging ellipsometry biosensor, setup its detecting standard curve (concentration - gray counts), and its detection limitation was 5.80 IU/mL, which can meet the clinical demands.2. The results of CA 15-3 concentrations detected in 80 serum samples by optical protein-chip and electrochemiluminescence showed no statistical significant (P>0.05), which indicated a good consistency. These results suggested the feasibility of this technique and its potential clinical diagnostic usage in the future.3. The difference of CA 15-3 concentrations between control and cancer groups was statistical significant (P<0.05), which was coordinate with their clinical situation.