Detection Of Multivariate Cancer Biomarkers By SERS Sensors Based On Paper Platform

Cancer is one of major public health problem worldwide and the second leading cause of death,placing a huge financial burden on families and societies.In 2022,the National Cancer Institute expects 1.918 million new cancer cases and 0.609 million cancer deaths.Lung cancer and prostate cancer are among the most common cancers in terms of morbidity and mortality.The early diagnosis of cancer is crucial to reduce the mortality of cancer.One of the important and effective ways is to detect the expression level of tumor markers.Due to the trace content of tumor markers in the early stage of cancer,it is urgent to develop a tumor marker detection platform with low cost,high sensitivity and strong specificity.Surface-enhanced Raman spectroscopy（SERS）is a common spectral detection technique,which can obtain 2～6 orders of magnitude enhanced Raman signal intensities by employing rough nanometallic surfaces,sometimes even up to 14 orders of magnitude,thus promoting the application of SERS in tumor markers detection.Paper-based analysis platform has attracted wide attention because of its advantages of low cost,good biocompatibility and fast analysis speed.Moreover,paper-based analysis platform can make fluid flow to specific areas by capillary action,which has great application prospects in the field of sensing analysis.In this thesis,a new paper-based SERS platform was established by combining paper substrate with SERS for the detection of multivariate tumour markers.The main research content of this thesis are described as follows:1.A paper-based SERS sensor with gold nanourchins（Au NUs）for the detection of cytochrome c（Cyt c）in non-small cell lung cancer（NSCLC）:Cyt c,a tumor marker of NSCLC,plays an important role in the diagnosis and therapy of NSCLC.A novel SERS sensor was constructed to detect Cyt c in the serum of NSCLC patients.The sensor was composed of Au NUs substrate,whose surface was functionalized with the Cyt c aptamer and the Cy5-labeled complementary DNA.In the existence of Cyt c,it could specifically bind to its aptamer,which led to the detachment of complementary strands modified with Cy5 and the great weakness of SERS signal.Cyt c in serum could be detected by the characteristic peak intensity of Cy5.Au NUs substrate possessed excellent SERS performance,which was strongly dependent on a large number of "hot spots" at the tips and between the nanogaps of aggregated Au NUs.SERS sensor based on Au NUs exhibited prominent reproducibility and specificity.The detection limit（LOD）of Cyt c in phosphate buffer was 1.148 pg/mL,while the LOD in human serum was 1.79 pg/mL.Serum samples from healthy subjects and NSCLC patients were detected by SERS sensor and enzyme-linked immunosorbent assay（ELISA）.It was confirmed that the SERS sensor could be used as a powerful tool for detecting Cyt c in serum.SERS sensor was expected to be applied in the practical clinical diagnoses of NSCLC.2.A SERS-lateral flow assay（LFA）test strip based on Au-Ag hollow nanoparticles（Au-Ag HNPs）for the detection of thrombin and platelet-derived growth factor-BB（PDGF-BB）in prostate cancer:In order to achieve rapid detection of multivariate tumor markers in clinical samples,a simple,convenient and ultrasensitive SERS-LFA test strip was prepared to detect two kinds of tumor markers associated with prostate cancer-thrombin and PDGF-BB.Au-Ag HNPs were modified by two Raman signal molecules（DTNB and NBA）with distinguishable characteristic peaks and detection aptamers,which were used as two labeled probes,while the capture aptamers were immobilized on two parallel test lines（T1 and T2）.When thrombin and PDGF-BB were present in the sample,a sandwich-structure complex formed by hybridization of targets,detection adapters and capture aptamers accumulated on the T lines,resulting in the color of T lines.Qualitative observation and quantitative analysis of thrombin and PDGF-BB were conducted through the location and color of bands,as well as SERS signal intensity.The contents of thrombin and PDGF-BB in plasma were detected by SERS-LFA strip in the range of 10 pg/mL and 10 μg/mL.The LODs of thrombin and PDGF-BB in human plasma were as low as 4.837 pg/mL and 3.802 pg/mL,respectively.SERS-LFA test strip had the advantages of great specificity,reproducibility and homogeneity.The expression levels of thrombin and PDGF-BB in plasma from prostate cancer patients and healthy subjects were detected by SERS-LFA test strip.The accuracy of SERS-LFA test strip was verified by ELISA.As a result,the SERS-LFA test strip had broad application prospects in the clinical diagnosis of major diseases like prostate cancer.3.A SERS-LFA test strip based on Au-Ag nanoshuttles（Au-AgNSs）for the detection of miR-196a-5p and miR-31-5p in lung cancer:To further improve the sensitivity and specificity of detection,a SERS-LFA test strip was constructed to detect miR-196a-5p and miR-31-5p with catalytic hairpin assembly（CHA）as a means of signal amplification.In this method,Au-Ag NSs were modified by biotinylated hairpin DNA 1（H1-bio）,which were used as two labeled probes.Two kinds of hairpin DNA 2（H2）were immobilized on two parallel test lines（T1 and T2）labeled with streptavidin.In the presence of target miRNAs,H1-bio on Au-AgNSs were opened,exposing biotin towards outside.CHA reaction occurred between target miRNAs,H1-bio and H2 immobilized on the T lines.The reaction products were captured on the T lines by the specific interaction of biotin and streptavidin,resulting in gray bands.At the same time,the released target miRNAs entered the next CHA cycle,causing the amplification of SERS signals.The location and SERS signals intensity of the bands reflected the type and content of target miRNAs in the samples.The prepared SERS-LFA test strip could detect miR-196a-5p and miR-31-5p with great reproducibility,high specificity,excellent homogeneity and super sensitivity.The whole detection time was short.The LODs of miR-196a-5p and miR-31-5p were as low as 1.171 nmol/L and 2.251 nmol/L in phosphate buffer,meanwhile 1.681 nmol/L and 2.603 nmol/L in human serum,respectively.Finally,SERS-LFA test strip was applied to detect miR-196a-5p and miR-31-5p in the clinical serum from lung cancer patients with different stages and healthy subjects.The expression levels of these two miRNAs in serum of lung cancer patients were significantly higher than that of healthy subjects.With the development of lung cancer,the expression levels of target miRNAs gradually increased.The designed SERS-LFA test strip based on CHA signal amplification provided a new method for miRNAs,proteins and other tumor markers research in the field of biomedical diagnosis,which was of vital importance in the early detection and postoperative monitoring of lung cancer.