Multiple SERS Immunoassay Based On Hydrogel Photonic Crystal Encoded Microspheres

The detection of tumor markers is of great practical significance for early cancer screening and disease assessment and diagnosis.In order to detect tumor markers,most of the current research focuses on the detection of a single marker,however,the complexity of the carcinogenesis process makes many tumor markers not specific to one cancer.Therefore,the development of methods to detect multiple tumor markers simultaneously has attracted extensive attention of researchers.The reported methods include radioimmunoassay,enzyme-linked immunosorbent assay,fluorescence immunoassay,chemiluminescence immunoassay,electrochemical immunoassay,and the like.Surface-enhanced Raman scattering(SERS)has many advantages,such as high detection sensitivity,narrow peak spectrum,stable signal,and insensitivity to light quenching and bleaching,and can be used for multiplex biological detection.As far as the current research situation is concerned,multiplex detection based on SERS technology is usually realized by using multiple Raman marker modes and Raman imaging.Multiple Raman marker modes require different label molecules,which reduces multiplex detection.The analytical throughput of the Raman imaging instrument is expensive,which limits its practical application.Photonic crystal coding is a new coding method.Inverse opal structure hydrogel photonic crystals have similar optical properties to photonic crystals,and the coding amount is huge,which can meet the requirements of high-throughput detection.Compared with crystals,its biocompatibility is better,and its porous structure can immobilize more probe molecules.Therefore,it will be a very meaningful research direction to develop microspheres encoded by inverse opal structure hydrogel photonic crystals as carriers,combined with SERS technology,to realize the discrimination and ultrasensitive detection of multiple tumor markers.The specific research contents of this paper are as follows:(1)Two kinds of antiopal hydrogel photonic crystal microspheres encoded by different reflection peaks were prepared by template replication method,and the CEA and AFP antibodies were modified respectively as solid phase immune substrates.At the same time,Raman probe molecules and antibodies were modified onto Ag nanoparticles to prepare SERS immune probes.The base probes prepared above were added into the two components of CEA and AFP at the same time to form sandwich structure.Using the reflection peak characteristics of microspheres and the characteristic color encoding and decoding under polarized light microscope,the microspheres were placed under Raman spectrometer to detect SERS signal and determine the concentration of the antigen to be measured,which successfully achieved the clear differentiation and simultaneous detection of multiple tumor markers.The prepared hydrogel photonic crystal microspheres have the advantages of stable coding,good biocompatibility and large specific surface area.The proposed SERS detection method for multiple tumor markers was simple to operate,distinct,stable and sensitive.The detection range of CEA and AFP were 1.0 × 10-9-1.0 × 103 ng/mL and 1.0 × 10-7-1.0 × 103 ng/mL,respectively.The detection limits were 4.2 ×10-10 ng/mL and 8.1 × 10-8 ng/mL,respectively.(2)The simultaneous detection of multiple SERS tumor markers was realized by combining the codec performance of hydrogel photonic crystal microspheres with the catalase mimicase performance of Au nanoparticles and using the signal amplification method of mimicase.First of all,Au nanoparticles themselves could be used as SERS base to improve the sensitivity of SERS detection.In addition,in the presence of H2O2,the Au nanoparticles played the role of Peroxidase mimicase to oxidize the inactive Raman signal molecule TMB into active TMBox,which enhanced SERS activity and further enhanced the sensitivity of SERS detection.By using the reflection peak position of hydrogel photonic crystal microspheres and the characteristic color decoding under polarizing microscope,and then placing the microspheres under the laser Raman spectrometer to measure the Raman signal,the accurate differentiation and quantitative detection of mixed antigens were realized.The SERS detection method for multiple tumor markers had high sensitivity and good stability,and was basically consistent with the actual clinical detection results.The detection ranges of CEA and AFP were 1.0 × 10-4-1.0 × 103 ng/mL and 1.0 × 10-3-1.0× 103 ng/mL,respectively,and the detection limits were 4.6 × 10-5 ng/mL and 7.0 ×10-4 ng/mL,respectively.(3)The SERS immunoassay of multiple tumor markers was performed by combining the codec performance of hydrogel photonic crystal microspheres with tyramine signal amplification strategy.Firstly,two hydrogel photonic crystal encoded microspheres were modified with CEA and AFP coated antibodies respectively,then the mixed antigens of CEA and AFP were added,and finally horseradish peroxidase labeled antibodies(HPR-CEA,HPR-AFP)were added to form solid phase sandwich immune substrate structure.In the presence of H2O2,Au@Ag nanoparticles used for tyramine labeling could be enriched by HRP catalytic deposition,thus forming a large number of SERS hot spots.Finally,the reflection peak position of the hydrogel photonic crystal microspheres and the characteristic color decoding under the polarizing microscope were used to measure the Raman signal of the microspheres under the laser Raman spectrometer,so as to achieve the accurate differentiation and ultra-sensitive detection of the mixed antigen.The SERS immunoassay method for multiple tumor markers showed obvious differentiation,strong anti-interference,high sensitivity and stability,which was basically consistent with the clinical detection results.The detection limits of CEA and AFP were 1.0×10-7-1.0×103 ng/mL and 1.0×10-5-1.0×103 ng/mL,respectively,and the detection limits were 1.7×10-8 ng/mL and 5.9×10-6 ng/mL,respectively.