Multiple SERS Biological Detection Based On Raman Photonic Crystal Beads

Surface-enhanced Raman scattering(SERS)spectroscopy has become popular because of its advantages of high sensitivity,good selectivity and strong anti-interference.The application of SERS in drug analysis,biological detection and other fields is very popular.In addition,multivariate detection technology makes it possible to detect multiple biomolecules at the same time,thus saving a lot of analysis time,cost and sample consumption.Photonic crystal beads is a kind of material with three-dimensional ordered structure,which is often used as a coding carrier for multiple detection.Therefore,the development of high sensitivity,multi-component SERS biological analysis with photonic crystal encoded beads as the carrier will be a very meaningful research direction.In this paper,we took silica photonic crystal as the solid phase immune base,coded with different reflection peaks,and combined with SERS signal,and designed three ways of amplifying Raman signal to improve sensitivity and realize multivariate detection.Specific research contents are as follows:Two encoded photonic crystal beads(reflection wavelength of 500 nm and 600 nm respectively)were conjugated with CEA and AFP antibodies to obtain solid phase immune substrates.Then we synthesized porous gold and silver alloy nanoparticles(p-AuAgNPs)with high density "hot spot" to prepare the Raman signal amplification probe.Finally,the two were added to the two-component antigens(AFP and CEA),and they reacted at 37?for about 30 min to form the sandwich composite structure of "solid phase antibody-tested antigen-Raman signal amplification probe".The type of antigen to be tested was determined by detecting the reflection spectrum of the beads,and the concentration of the antigen to be tested was determined by detecting its Raman signal.The detection limits of CEA and AFP were 1×10-9 and 1×10-8ng/mL,respectively.The new multi-component detection method we proposed is characterized by stable coding,low cross interference and high sensitivity.Two encoded photonic crystal beads(reflection wavelength of 500 nm and 600 nm respectively)were conjugated with CEA and AFP capture antibodies to obtain solid phase immune substrates.Then the corresponding CEA and AFP binomial antigens were added.Then AuNPs modified with CEA and AFP antibodies were added and reacted for 30 min at 37? to form the sandwich immune structure.Among them,AuNPs had dual SERS signal enhancement function:(1)as SERS base,it could enhance the signal of Raman report molecule(TMB);(2)AuNPs has the performance of catalase mimicking enzyme.In the presence of H2O2,it will convert inactive Raman reporting molecules into active Raman reporting molecules,thus enhancing the signal.Therefore,after the formation of sandwich immune structure,H2O2 and inactive Raman reporter molecule(TMB)were added and activated at 37? for about 20 min.The inactive TMB was oxidized to active TMB.Raman signal was immediately detected and the antigen concentration was determined.Finally,the types of antigens to be tested were determined by detecting the reflectance peak spectrum of the beads.The detection limits of two tumor markers,CEA and AFP,were 10-3ng/mL and 10-2ng/mL,respectively.The new multi-component detection method is expected to be a good tool in low cross interference,simple operation and low costTwo encoded photonic crystal beads(reflection wavelengths of 500 nm and 600 nm,respectively)were conjugated with CEA and AFP antibodies to obtain solid phase immune substrates.Then the corresponding CEA and AFP binomial antigens were added.Then horseradish peroxidase-labeled CEA and AFP antibody(HPR-CEA,HPR-AFP)were added and they reacted at 37? for about 45 min to prepare the sandwich immune structure.In the presence of H2O2,horseradish peroxidase can convert tyramine into an intermediate that forms covalent compounds with aromatic amino acids on the protein surface and binds to the surface of the sandwich immune structure.By labeling the tyramine with SERS tags in the form of cubic Au@Ag nanoparticles functionalized with a Raman-active probe(4-MBA),cubic Au@AgNPs@4-MBA was deposited and aggregated near the proteins,so the SERS signal of 4-MBA could be detected and amplified.The type of antigen to be tested was determined by detecting the reflectance peak spectrum of the beads,and the concentration of the antigen was determined by detecting its Raman signal.The lowest concentrations of CEA and AFP we can detect are 10-5 ng/mL.The SERS signal measured by the tyramine signal amplification had low cross-interference,high sensitivity and was about 4 times as strong as the traditional SERS immune analysis.