A Novel Surface Enhanced Raman Scattering Nanoprobe Based Detection Method For Pheochromogranin A

Neuroendocrine tumors（NENS）are relatively rare and highly heterogeneou s tumors.Although its biological behavior and clinical diagnosis and treatment have been explored for more than 100 years,its pathogenesis is still not very clear.Moreover,the symptoms and signs of NENS are not typical,and the cli nical manifestations are diverse,so most of the patients with NENS are found in the late stage,and they have lost the chance of radical cure due to local di ffusion and distant metastasis at the time of diagnosis.CGA is the most comm only used and effective tumor marker in the auxiliary pathological examination of neuroendocrine tumors,which is used to guide the treatment and evaluate t he curative effect.When the serum CGA level is higher than 94ng/ml,it indic ates the risk of cancer.CGA is the best serum or plasma marker for the diagnosis and treatment of general neuroendocrine diseases.It has become the main development directi on of diagnosis and treatment of neuroendocrine tumors to develop new ultra s ensitive diagnostic methods for chromogranin A,an important diagnostic marke r of the disease.CGA can detect tumors before symptoms appear.Their use as screening agents in patients with diffuse abdominal symptoms can reduce the age of tumor detection and improve the cure rate of surgery.However,limited by the sensitivity of detection methods,only about 60-80%of NENS patients can detect high levels of CGA.Surface enhanced Raman spectroscopy（SERS）is a rapid and non-destructi ve detection technology,which can identify and quantify biological molecules（l ipids,proteins,DNA,etc.）with obvious chemical and molecular characteristics.In addition to molecular specificity,Raman scattering can be used for real-time detection due to its compatibility with near-infrared excitation source,which makes it widely used in biosensor field,such as in vivo imaging.In this stud y,we use SERS technology,through the design and synthesis of a new Rama n probe to detect the endocrine tumor marker chromogranin A（Cg A）,to achie ve the homogeneous,sensitive and rapid detection of endogenous tumor marker s,and provide a research basisfor the future application of SERS technology i n cancer screening.Firstly,the SPA domains SPAZ^MM、SPAZ^DM and SPAZ^TM,which can spec ifically recognize the Fc segment of Ig G antibody,were designed and synthesi zed to realize the directional connection of Ig G antibody.Then,different antibo dies that recognize the same antigen（Cg A）were modified on Ag NPs and MN s with Raman reporter and linker to form two probes.Because of the regular orientation of the antibodies on the surface of the Raman probe,more sensitive Raman signals can be detected when the modified antibody is directionally cou pled to linker.In the presence of chromogranin A,the signal of the reporter m olecule on the silver probe was greatly enhanced due to the proximity of the t wo probes due to the recognition of the antibody to the protein,otherwise,on ly the weak signal of the reporter molecule itself was obtained.By detecting t he intensity of Raman signal,we can quickly judge whether Cg A exists or not.In this study,we developed a new solid-phase modification method and o btained a new linker.The experimental results showed that spa domain mutants SPAZ^MM,SPAZ^DM and SPAZ^TM had strong recognition ability for the Fc segme nt of Ig G antibody;In the preparation process of nanomaterials,it is found tha t the modification of SH-PEG₅₀₀ are very important for the stability of the pro be and the enhancement of Raman signal.On the basis of the above technolog ical progress,we have successfully sensed the biological signal of antigen anti body recognition by using SERS technology.The detection limit is as low as1ng/ml,which is far lower than the common clinical detection demand（20ng/m L）,and higher than the detection limit of 8ng/m L reported in academic articl es.A homogeneous micro ultra sensitive method for the detection of Cg A was established.