Construction Of SERS-LFA Test Strips And Its Application In Tumor Marker Detection

Cancer is currently a persistent public health challenge shared globally and is expected to be the most significant barrier to life expectancy growth in the 21st century.Currently,there is a high incidence and mortality rate for both lung cancer and head and neck squamous cell carcinoma.Early diagnosis of cancer is critical to reduce cancer mortality,and one effective way to do this is to test the expression levels of tumor marker.Tumor markers are widely used for early diagnosis and prognosis assessment of cancer because they can reflect specific biological changes during tumorigenesis and progression.Since the expression level of tumor marker in the early stage of cancer is very low and one tumor marker is often associated with multiple cancers,a highly sensitive and specific as well as efficient tumor marker detection platform is of great significance for the early diagnosis and treatment of cancer.Surface-enhanced Raman scattering(SERS)is a fast and non-destructive technique with quantitative detection,while lateral flow assay(LFA)strips are simple,inexpensive,portable and efficient.Catalytic hairpin assembly(CHA)is a non-enzymatic signal amplification strategy with low background signal,which can improve the sensitivity and specificity of the assay.In this study,two novel SERS-LFA test strips were constructed for the rapid,highly sensitive and specific detection of tumor markers using CHA as the signal amplification strategy,SERS as the quantitative analysis technique and LFA strips as the detection platform.The main studies are as follows:1.SERS-LFA strip based on Gold nanocages(GNCs)for the detection of miR-21 and miR-196a-5p in none small cell lung cancer(NSCLC):In this method,two SERS probes were prepared by modifying two hairpin DNA sequences 1(Hairpin DNA 1 labeled with biotin,H1-bio)on GNCs.Two hairpin DNA sequences 2(H2)and streptavidin(SA)were immobilized on the test line(T line)of the test strip.When target miRNAs(miR-21 and miR-196a-5p)is added and flowed to the position of SERS probe located on the binding pad,the target miRNAs will hybridize with the H1-bio,and the H1-bio hairpin modified on the GNCs will open while exposing the biotin molecules.The complex formed by target miRNAs with H1-bio flows to the T line and hybridize with H2 on T line to form H1-bio-H2 while releasing the target miRNAs.The specific binding of exposed biotin to SA allows the reaction complex to be immobilized on the T line,showing a gray band.The expression levels of miR-21 and miR-196a-5p in the samples were obtained from the intensity of the characteristic peaks located at 1337 cm1 and 1594 cm-1 on the T line.The limit of detection(LOD)of miR-21 and miR-196a5p in human urine were 3.31 pmol/L and 2.18 pmol/L,respectively.The detection time was only 30 min.Finally,the quantitative analysis of miR-21 and miR-196a-5p in human urine samples was successfully achieved by this method,and based on the expression levels of the two target miRNAs,NSCLC patients and healthy individuals can be precisely distinguished.2.High-throughput SERS-LFA strip based on Pd-Au core-shell nanorods(Pd-Au NRs)for the detection of TP53 and PIK3CA E545K in head and neck squamous cell carcinoma(HNSCC):In order to further improve the detection efficiency,a highthroughput SERS-LFA test strip integrating eight test strips was constructed based on the previous test strip.The number of each strip corresponds to the number of the sample to be tested,and the expression level of circulating tumor DNA(ctDNA)of each sample can be obtained by reading the intensity of the characteristic peak on the T line of each strip,respectively.This high-throughput SERS-LFA strip has a low LOD of pmol/L for TP53 and PIK3CA E545K in human serum with a linear dynamic response range of 1 pmol/L-10 μmol/L.The ability of the high-throughput SERS-LFA strips to differentiate healthy individuals from HNSCC patients was confirmed by testing the actual samples,and the accuracy of the high-throughput SERS-LFA strips was also verified by real-time quantitative polymerase chain reaction(qRT-PCR)results.This method has the potential for early diagnosis of HNSCC.