Fabrication And Application Of Lateral Flow Assays With Ultrasensitive And Multiplex Detection

With the increasing demand for accurate diagnosis of infectious diseases,cardiovascular disease and cancer,as well as the rapid growth of the“Internet plus”,the traditional detection method cannot meet the clinical requirement of“getting accurate test results in the shortest time”.So,the simple,fast,miniaturized,and highly sensitive point of care testing?POCT?has been widely concerned by the public.Lateral flow assay?LFA?is the most widely used POCT technology owing to its simple operation,rapid detection,and robustness in various applications.However,the current LFA shows low sensitivity,low detection throughput,long detection time,and narrow linear dynamic range?LDR?.All these technical bottlenecks seriously restrict the technical innovation of POCT.Surface enhanced Raman scattering?SERS?has been widely used in analytical chemistry,biomedicine,food safety,and environmental hygiene monitoring and other fields,because of its high sensitivity,no sample pretreatment,simple operation,high detection speed,high accuracy,and no destruction of the sample to be tested.In this paper,we developed the SERS nanotags based LFA?SERS LFA?and applied it in multiplex detection of biomolecules.The main contents and results are as follows:?1?The silver-gold core-shell bimetallic nanoparticles Ag^MB@Au,Ag^NBA@Au,and Ag^R6G@Au were synthesized successfully to form SERS nanotags,the Raman dyes?RDs?embedded in the interior gap between Ag and Au.The SERS nanotags has stable coding ability and highly enhanced Raman signal.Under the excitation of a 785 nm laser,the SERS nanotags with the radius of silver core and the thickness of gold shell of 5 nm has the strongest Raman signal.Moreover,different SERS nanotags can be distinguished directly according to their raw Raman characteristic peaks without special algorithm.Simulation results by FDTD show that with the decrease of gap size and gold shell thickness,the LSPR absorption of the SERS nanotags shows a red shift.Taking the red-shifted absorption peak as the wavelength of excited light,the maximum ratio of near field intensity between the interior gap and the outer gold shell was obtained.The theoretical calculation results got by FDTD simulation are consistent with the experimental results when a 785 nm laser is used.This provides a theoretical basis for further improving the signal sensitivity of SERS nanotags,and has important guiding value in practical SERS nanotags based multiplex biomolecular detection.?2?A core-shell SERS nanotags(Ag^NBA@Au)based LFA for multiplex and quantitative detection of cardiac biomarkers has been prepared to realize the early diagnosis of acute myocardial infarction?AMI?.In practice,three test?T?lines were immobilized on the nitrocellulose?NC?membrane for the detection of Myo,cTnI,and CK-MB,respectively.The limit of detection?LOD?for Myo,cTnI,and CK-MB were calculated to be below the clinical cutoff values,which were 3.2,0.44,and 0.55 pg mL^-1,respectively.Due to the amplified signal of the SERS nanotags and the high surface area to volume ratio?SVR?of porous NC membrane,The LDR of Myo,cTnI,and CK-MB were 0.01⁵00 ng mL^-1,0.01⁵0 ng mL^-1,and 0.02⁹0ng mL^-1,respectively,which cover the clinical range of Myo,cTnI,and CK-MB in human body.The results got from SERS LFA are comparable to the Pure Food and Drug Administration?FDA?approved chemiluminescence immunoassay?CLIA?method.Compared to CLIA,SERS LFA has the merits of ultra-sensitivity,rapid detection time,low-cost,easy to use,free of sample pretreatment and professional labors.?3?A novel LFA based on RDs encoded core-shell SERS nanotags(Ag^MB@Au,Ag^NBA@Au and Ag^R6G@Au)for rapid quantification of three cardiac biomarkers on a single T line has been proposed.The LOD for CK-MB,cTnI,and Myo were calculated to be 0.93,0.89,and 4.2 pg mL^-1,respectively,which were below the cutoff values in human body.The LDR of CK-MB,cTnI,and Myo were 0.01⁵00 ng mL^-1,0.01⁵0 ng mL^-1,and 0.02⁹0 ng mL^-1,respectively,which cover the clinical ranges of the three biomarkers.With the spatial separation presented in the three T lines SERS LFA substituted by the wavelength separation based on encoded SERS nanotags in the single T line SERS LFA,the detection time?17 min?was greatly decreased about three times,which is particular favorable for quick diagnosis of sudden diseases such as AMI to reduce mortality.The single T line SERS LFA need less reagent consumption,decreased preparation time,easy experiment operation.We envision this method to find wide applications in in vitro diagnostics?IVD?.?4?A novel lateral flow microarrays?LFM?based on RDs encoded core-shell SERS nanotags(Ag^MB@Au and Ag^NBA@Au)for ultrasensitive quantification of eleven nucleic acids of pathogens for early and accurate diagnosis of respiratory tract infection?RTI?has been developed.The 2×3 microarray was set on the NC membrane,each T dot immobilized two capture nucleic acid.The rapid quantification of influenza A,influenza B,parainfluenza 1,parainfluenza 2,parainfluenza 3,adenovirus,respiratory syncytial virus,chlamydia pneumonia,coxiella burnetil,mycoplasma pneumonia,and legionella pneumophila was realized within 20min.Owing to the signal amplification of encoded SERS nanotags and high SVR of NC membrane,the LOD for the pathogen nucleic acids were lower than 0.041 pM.The LDR of measurement of the target nucleic acids of the eleven RTI pathogens were 1 pM⁵0 nM,which span 5 orders of magnitude.This novel approach could be widely adopted in the early and precise diagnosis of RTI and other diseases.