Multiplex Protein Sensing Chip Based On Surface Enhanced Raman Spectroscopy(SERS)

As the expression level of proteins in human body is closely related to the occurrence and progression of cancers,the development of highly sensitive,high-throughput,rapid and low-cost optical technology for multiplex protein detection is of great significance to the fundamental research in oncology and the clinical applications in early cancer diagnosis and therapeutics.This paper focuses on the development of a novel multiplex protein detection platform which combines surface enhanced Raman spectroscopy(SERS)technique with the microfluidic chip.This study aims to improve the detection sensitivity,multiplexing ability,efficiency as well as to reduce the overall costs.Here,the spectral-spatial joint encoding method has been proposed to develop a novel analytical platform for high-throughput protein detection,which is further employed for the detection of tumor markers and the study on the interaction mechanism of anti-cancer drugs.This platform provides a new technological route for the early cancer diagnosis and therapeutics.The main innovations of this thesis are listed as follows:(1)Gold@silver core-shell nanorods were introduced in SERS-based immunoassays to improve the sensitivity of the detection.First,gold@silver core-shell nanorods were fabricated by coating a silver shell onto the gold nanorods,and the SERS activity of the nanoparticles was also studied.Then,the SERS immune probes were achieved by labeling the Raman reporters and the antibodies onto the surfaces of the nanoparticles.Finally,the SERS probes were employed for immunoassays,in which the specificity and sensitivity were evaluated.The experimental results showed that the sensitivity of the SERS-based immunoassay with gold@silver core-shell nanorods can be improved by 4 orders of magnitude compared to that with gold nanorods.(2)SERS spectral encoding technique was developed for multiplex protein detection.In the experiment,two kinds of immune probes with different Raman signatures were prepared by labeling gold@silver core-shell nanorods with two kinds of Raman molecules and two different antibodies.These two immune probes were used to identify two different proteins respectively.Here,the simultaneous detection of two tumor biomarkers,p53 and p21,was performed in a single immunoassay using SERS encoding technique,which avoids the repetitive operation,reduces sample and reagent consumption,and fully exploits the advantages of SERS in multiplex detection.(3)A novel SERS spectral-spatial joint encoding method was proposed.Based on the idea of joint encoding,a SERS-assisted 3D barcode chip has been designed for multiplex protein detection.In the detection process,a 2D protein hybridization array was constructed with microfluidic channels,and then the SERS probes was employed to simultaneously detect the multiple proteins in all of the samples.By loading the SERS spectral information into a 2D array,a SERS-assisted 3D barcode is created,which combines the microfluidics-based 2D spatial coding with the SERS spectral coding.Through the decoding of the 3D barcode,the information about different proteins in all of the samples could be obtained.The platform shows the a.dvantages of large information storage capacity,high sensitivity(～10 fg/mL),fast response(30 min)and simple operation,which provides a new route for high throughput biosensing.(4)A versatile microfluidic platform for the detection of tumor secretions was presented,in which the mechanism of anti-cancer drugs and the process of intercellular communication were studied.In this design,two cell culture chambers and a set of pneumatic microvalves were integrated with the SERS-assisted barcode chip to develop a fully integrated and highly automated platform which enables the on-line detection of cell secretions,the monitoring of cancer-immune intercellular communication and anticancer drug screening.By exploiting the advantage of SERS-microfluidic chip in multiplex detection,this platform shows the advantages of low cost and high degree of automation,which is expected to provide a powerful tool for the discovery of new anticancer drugs.