Multiple Vertical Flow Immunoassay Based On SERS Nanotags

With the improvement of people's living standards,more and more attention is paid to medical and health.People expect to obtain accurate diagnostic information related to diseases through simple and easy-to-use detection methods.However,large immunoassay equipment is usually used in the actual clinical diagnosis,although its detection sensitivity can reach pg m L^-1level,the detection process is cumbersome,requiring professional operators,and the purchase and maintenance cost of the instrument are high.In the 1970s,as the development of colloidal gold labeling technology,two classical immunological detection methods,vertical flow immunoassay(dot gold immunofiltration)and lateral flow immunochromatography were developed.Among them,vertical flow immunoassay(VFA)has the advantages of simple structure,easy preservation and portability,fast and direct interpretation of detection results,as well as simultaneous detection of multiple targets without hook effect.The advantages of VFA makes it gradually become the technical basis for the development of point of care testing(POCT)diagnostic kit.At present,most of the commercial VFA kits are based on single biomolecule detection,which can obtain limited diagnostic information and low sensitivity.In order to obtain high-throughput biomolecules detection,people usually use fluorescent or quantum dots to encode the detected substances.However,they are not stability and easy to photobleaching,moreover quantum dots have biological toxicity,which limits their application.Since 1974,high-quality Raman spectra of single molecular layer pyridine molecules on the surface of rough silver electrode was found by Fleischmann.The study of surface enhanced Raman scattering(SERS)spectra has attracted more and more attention.SERS has been widely used in analytical chemistry,biomedicine,customs quarantine,food safety and environmental health monitoring,due to its high sensitivity,no sample pretreatment,no interference from aqueous solution,multi-coding,simple operation,fast detection speed,high accuracy and no damage to the samples to be tested etc.Therefore,this paper carried out the construction of multiple VFA system based on SERS nanotags for biomolecules detection.The main contents and conclusions of this paper are as follows:(1)A VFA system based on gold core silver shell SERS nanotags was developed for the detection of prostate specific antigen(PSA),carcinoembryonic protein(CEA)and alpha fetoprotein(AFP).Firstly,taking the synthesis of Au^4-MBA@Ag as an example,the concentration of silver nitrate was optimized.Then three kinds of Raman dyes were embedded into the internal gap between gold core and silver shell nanoparticles,and Au^NBA@Ag,Au^4-MB@Ag,and Au^4-NBT@Ag SERS nanotags were successfully prepared.Then,the VFA kit was assembled for the detection of PSA,CEA and AFP,and the limits of detection(LOD)was calculated to be 0.37,0.43 and 0.26 pg m L^-1,respectively.For the detection of PSA,CEA and AFP in serum,the recoveries were 96.2%-108.5%,90.1%-111.4%and 91.6%-105.1%,respectively,indicating the high stability and excellent reproducibility of the biosensor.(2)A VFA system based on nanofluid channel array and gold core silver shell SERS nanotag was constructed for the detection of C-reactive protein(CRP),interleukin-6(IL-6),serum amyloid protein(SAA)and procalcitonin(PCT).Four kinds of Raman dyes encoded SERS nanotags were prepared(Au^NBA@Ag,Au^4-MBA@Ag,Au^DNTB@Ag,and Au^MB@Ag)to detect inflammatory biomarkers in a single test of the 2×2 array.The detection limits of CRP,IL-6,SAA and PCT were calculated to be 53.4,4.72,48.3 and 7.53 fg m L^-1,respectively.In addition,the linear dynamic ranges for CRP and IL-6,as well as SAA and PCT were 0.01-1000 ng m L^-1 and 0.001-1000 ng m L^-1,respectively,which could mostly cover the clinical concentration range.This method has potential clinical diagnostic value,and is expected to provide technical support for rapid and accurate screening of infectious inflammation in POCT scene.(3)A VFA system based on ordered pore substrate and gold core silver shell SERS nanotag was constructed for the detection of multiple food toxin factors such as ochratoxin(OTA),aflatoxin(AFB1)and tetracycline(TC).Firstly,three kinds of SERS nanotags were prepared(Au^NBA@Ag,Au^4-MBA@Ag,and Au^DNTB@Ag),and they were used to labell OTA,AFB1 and TC,respectively.Then,the concentration of nitrocellulose solution was optimized during the preparation of ordered substrate.In addition,by using five different sizes of silica particles as the etching template,the position of the reflection peak of the prepared ordered membrane is optimized.It is concluded that the best result can be obtained when 785 nm laser was selected as excitation source and reflection peak of 632.4 nm ordered membrane was selected as detection substrate.Moreover,the Raman enhancement mechanism is also discussed.The LOD values of OTA,AFB1 and TC were 8.2,13.7 and47.6 fg m L^-1 respectively,which were better than those of traditional nitrocellulose membrane.This study provides a theoretical basis for further improving the sensitivity of multiplex biomarkers detection,and has important guiding value in the actual application.