Detection Of Carcinoembryonic Antigen By Scanning Electron Microscopy With Nano-gold Probe Assisted Antibody Chip

For decades,malignant tumors become a great threat to human health,and early disease diagnosis is the key to cure cancer.The emergence of tumor markers has brought more possibility for the early diagnosis of the tumor.Carcinoembryonic antigen(CEA),which is a glycoprotein,is one of the most common tumor markers present in colon cancer and embryonic colonic mucosal epithelial cells,and has been widely used for the diagnosis and monitoring of various digestive tumors.At present,CEA and major of other antigens and antibodies in clinic are still detected using traditional enzyme-linked immunosorbent assay(ELISA).ELISA has the advantages of easy operation and pM sensitivity.However,the ELISA is a little time-consuming and only relatively quantitative,which means that one have to use a serial standard samples along with the samples to be tested to draw a standard curve that was used for quantifying the concentration of interest of the sample.This type of methods that need standard curve to evaluate test samples are unable to provide an absolute number of molecules of interest of molecules.In the beginning of this study,we were supposed to get one or a few peptides with high affinity to CEA antigen by using phage library screening technology so that we can construct a stable gold nanoparticle(GNP)probe modified with CEA-binding peptides.Then we further develop a biochip-based SEM counting system that captures CEA taget molecules in a sandwich format by CEA-binding peptides functionalized GNP pobe and CEA antibodies anchoring chip,and can count the number of GNP probes on chip with SEM and counting softwares.The rapid capture and detection of CEA antigen can achieve by this design at the range of concentration of CEA.Thus,one can directly read the number of CEA moleculars in a sample with the method described above if we can successfully get the designed GNP probe.In this study,M13 phage loop peptide library was used to screen the peptides that have high affinity with CEA.However,we failed to obtain the ideal peptides which are specially attached to CEA covered on the surface of well-plateafter through multiple screening experiments.We think that the screening efficiency with phage library to get short CEA-affinity peptites is very low and it is still a challenge to get a certain peptide that has the specific affinity to target molecules by phage screening technology.In the future we need to develop a new phage screening system if we would like to readly use phage display for exploring interaction between the molecules.Regarding the above results,we redesigned a gold nanoparticle(GNP)probe-assisted sandwich immunoassay that rely on a GNP probe with CEA antibodies,monoclonal antibodies covered silicon chip and Scanning Electron Microscope(SEM)employed to"count" protein molecules captured by GNP and chip.The proof-of-concept demonstration of counting carcinoembryonic antigen(CEA),as the model cancer protein biomarker,shows that our strategy is untral-sensitive(limit of detection is down to-4 fM,about 1000 times sensitive than ELISA)and able to absolutely quantify CEA molecules in samples.The chip-based sandwich immunoassay allows for counting antigen molecules in a tiny drop of solution due to use of nanotechnology and SEM technique,which can serve as a promising protein-quantitative platform in future clinical assay.