The Construction Of Raman And Terahertz Sensors Based On Silicon-based Chips And Their Application In Tumor Detection

Background:At present,tumor is still a serious threat to human health.The number of tumor deaths in the world is about 8.2 million every year,and the mortality rate is the second among common disease.Early screening and prognosis evaluation of tumor are helpful to improve the detection rate and enhance the confidence of patients in treatment,playing a key role in the diagnosis and treatment of tumor.Histopathological biopsy and imaging examination are the main means of tumor diagnosis in clinic.As the “gold standard” of diagnosis,histopathological biopsy is not suitable for large-scale screening because of its invasive nature.Imaging examination with high cost,is not suitable for large-scale development,and its accuracy is highly dependent on the personal experience of the operating physician.Because the detection of tumor markers is fast,simple,noninvasive and suitable for large-scale development,more and more tumor markers have been found,verified and used in large-scale screening,clinical diagnosis and prognosis evaluation of tumor diseases.In addition,with the rapid development of high-throughput sequencing and the establishment of omics,a large number of biomedical databases have been established.Combined with bioinformatics methods and technologies,it has become a necessary way to screen,evaluate and verify tumor markers.The detection technology of tumor and its markers is also constantly updated,among which the establishment of biosensor has created a new era of high sensitivity detection.Optical sensor is one of the most common biosensors,including surface plasmon resonance,fluorescence,optical fiber and other sensor types.In recent years,with the rapid development of vibration spectrum technology represented by Raman spectrum and terahertz(THz)spectrum,a research upsurge has been set off in the biomedical field,due to the advantages of fast,label-free analysis and so on.Because Raman spectrum and THz wave belong to different frequency band in electromagnetic spectrum,but there are some overlaps,and they belong to resonance scattering and absorption spectrum respectively,they have good complementarity in research and application.Therefore,surface-enhanced Raman scattering(SERS)and THz metamaterials sensors,as new optical sensors,have been widely used in the detection of tumor markers and cells.However,SERS and THz metamaterials sensors are not really used in clinical until now,mainly due to the preparation of high-quality SERS active substrate and effective contact with the target for SERS sensors.For THz metamaterials sensors,the dry detection environment is easy to affect the normal morphology and function of molecules,cells and tissues,so it is urgent to solve these problems in clinical application.To sum up,this study aims at the problems for screening of new tumor markers,and tumor detection by SERS and THz biomaterial sensors,using the bioinformatics analysis method,SERS active substrate,nanoparticles(NPs)and micro-nano structure.The correlation between tumor microenvironment and the prognosis of 20 kinds of malignant tumors was evaluated,and potential gene and protein tumor prognosis markers were screened out,providing a new idea for the screening of tumor markers.Furthermore,SERS aptsensor based on enhanced substrate and silver nanoprobe and THz sensor based on silicon-based metamaterials were constructed to detect protein markers with high sensitivity,specificity and stability,and characterize the activity and water content of tumor cells,respectively.This not only provides a new sensing method for the simple and rapid detection of tumor,but also lays the foundation for promoting the application in clinical practice.Objectives:This study uses bioinformatics analysis methods to evaluate the correlation between tumor microenvironment and the prognosis of 20 kinds of malignant tumors,and to explore potential tumor prognostic markers from the gene and protein levels.Furthermore,the SERS aptsensor based on enhanced substrate and silver nanoprobe was constructed to detect protein markers with high sensitivity,specificity and stability.The THz sensor based on silicon-based metamaterials was constructed to characterize the activity and water content of tumor cells.They provide a new idea for the screening of tumor markers and a new way for the detection of tumor related proteins and cells with a sensitive,specific,stable,simple and rapid sensing method.Materials and Methods:1.Bioinformatics analysis of prognosis related markers of various malignant tumors based on tumor microenvironment(1)By using TCGA database and ESTIMATE algorithm,the correlations between stromal score and immune score and prognosis of 20 kinds of common malignant tumors were analyzed,and the related tumor types were selected and classified into stromal and immune score group,respectively.(2)The common differentially expressed genes of tumors in stromal score group and immune score group were screened,and the functional enrichment analysis was carried out.(3)Verify the relationship between the common differentially expressed genes of stromal and immune score group and the prognosis of related cancer,as well as the difference of expression in normal and tumor tissues.2.Evaluation of five commercial SERS active substrates and preparation of metal NPs(1)The characterization of five commercial SERS active substrates,including the surface morphology by common optical microscopy and scanning electron microscopy(SEM)and spectrum by Raman imaging spectrometer.(2)Five kinds of commercial SERS active substrates were evaluated comprehensively by three kinds of Raman labeled molecules,including the optimum excitation wavelength,detection repeatability and enhancement effect,to screen the best SERS substrate(3)Three kinds of metal NPs were prepared and characterized,including two kinds of gold nanoparticles(AuNPs)with different diameters and one kind of silver nanoparticles(AgNPs).3.Detection of IGF-?R protein by SERS aptsensor constructed with enhanced substrate and silver nanoprobe(1)Construction of SERS aptsensor based on aptamer modified Q-SERS substrate and silver nanoprobe: optimization of the reaction system between insulin-like growth factor 2 receptor(IGF-?R)protein aptamer and DNA probe,preparation and screening of SERS nanotag,and modification of Q-SERS substrate by aptamer.(2)The performance evaluation of SERS aptsensor for IGF-?R protein detection,includes sensitivity,specificity and stability evaluation.(3)Preparation of serum samples containing standard IGF-?R protein solution and detection by SERS aptsensor.4.Preparation,simulation and evaluation of silicon-based THz metamaterials(1)Verify the detection ability of THz time-domain spectrometer for cell suspension.(2)Design and prepare the silicon-based THz metamaterials of split-ring resonator(SRR)array,composed of five concentric square metal rings with four gaps.(3)By using COMSOL software,according to the finite element analysis,the resonance behavior and the surface electric field distribution of the metamaterial at its resonance frequency were simulated,and the sensitivity to the change of surface dielectric constant was simulated.(4)The secretion of cytokines,including IL-6,IL-8,GM-CSF and GRO?,were detected in the cell supernatant collected from Petri dish with and without THz metamaterials.5.Characterization of cell activity and water content based on metamaterial-enhanced THz spectroscopy(1)The effects of U87 cells on the resonance peak of THz metamaterials in different humidity(3% and 7%)were monitored continuously.(2)The repeatability of continuous monitoring of U87 cells was evaluated by THz metamaterials with different resonance peaks made from the same batch and structural parameters.(3)The thickness of U87 cells was examined by atomic force microscopy(AFM),and PBS liquid layer with equivalent cell thickness was detected by THz metamaterials.Results:1.Bioinformatics analysis of prognosis related markers of various malignant tumors based on tumor microenvironment(1)Stromal score or immune score can evaluate the prognosis of six tumor types,which are divided into immune score group including invasive breast cancer(BRCA),lung adenocarcinoma(LUAD),renal clear cell carcinoma(KIRC),low-grade brain glioma(LGG)and skin melanoma(SKCM),and stromal score group including gastric adenocarcinoma(STAD),LGG and SKCM.(2)116 and 54 common differentially expressed genes were screened out in stromal score group and immune score group respectively,and they were enriched to participate in biological process(BP),cell composition(CC)and molecular function(MF)with high consistency.In protein interaction(PPI),PTPRC and ITGAM are the most important central nodes for stromal and immune score group,respectively.(3)The expression level of 108 and 53 common differentially expressed genes in stromal and immune score group are related to the prognosis of at least one tumor in the group.There are significant differences in the expression of 79 genes between normal and tumor tissues.2.Evaluation of five commercial SERS active substrates and preparation of metal NPs(1)The substrates of Enspectr,Ocean-SP,Q-SERS,Ocean-Au and Ocean-AG substrates are glass,glass,silicon,paper and paper,respectively,on which metal NPs or thin sheets are distributed to enhance signal.The Raman signals of each substrate are obtained with the 532 nm and 785 nm lasers,but the difference between them is large.(2)The optimal excitation wavelengths of Enspectr,Ocean-AG and Ocean-SP substrates are 532 nm,but of Q-SERS and Ocean-Au substrates are 785 nm.The Enspectr and Q-SERS substrates have good detection repeatability,and the Ocean-AG and Q-SERS substrates have high enhancement effect.Therefore,the Q-SERS substrate is selected as the most suitable substrate to build sensors.(3)AuNPs at 23 nm and 32 nm and AgNPs at 20 nm were successfully prepared.They are spherical,uniform in size,scattered in distribution,without obvious aggregation and without obvious impurities in the background.The UV-Vis spectrum shows the unique absorption spectrum of AuNPs and AgNPs.3.Detection of IGF-?R protein by SERS aptsensor constructed with enhanced substrate and silver nanoprobe(1)The hybridization between IGF-?R protein aptamer and DNA probe can be completed in 10 minutes at room temperature.(2)The SERS nanotags constructed by AgNPs combined with Q-SERS substrate can produce the strongest SERS signal.(3)Only when the Q-SERS substrate is modified by aptamers,it can be combined with SERS nanotags.Therefore,the SERS aptsensor based on aptamers modified Q-SERS substrate and silver nanotags were constructed successfully.(4)SERS aptsensor enables quantitative detection of IGF-IIR protein,with the limit of detection of 141.2 fM,and has good specificity,intra assay repeatability and inter assay reproducibility.(5)The recoveries of standard IGF-?R protein in serum samples ranged from 97.1% to 106.0%,with RSD values of 1.1%-4.4%,which verified the feasibility and applicability of this SERS aptsensor in clinical detection.4.Preparation,simulation and evaluation of silicon-based THz metamaterials(1)THz time-domain spectrometer can quantitatively detect U87 cell suspension in a certain concentration range,and the relationship between cell concentration and THz absorption coefficient is linear under a certain frequency.(2)There are two resonance peaks of THz metamaterial in the range of 0.2-2 THz,which are located at 1.375 THz and 1.737 THz,respectively.Moreover,adherently growing cells can cause a decrease in transmission and red shift of resonant frequency.(3)The simulation results show that the low-frequency resonance is mainly caused by the outer ring,while the high-frequency resonance is caused by the middle ring.The low-frequency resonance peak is highly sensitive to the change of the surface dielectric constant of THz metamaterial.(4)THz metamaterial can significantly increase the content of IL-6,but significantly reduce the content of IL-8,GM-CSF and GRO? in the cell supernatant.5.Characterization of cell activity and water content based on metamaterial-enhanced THz spectroscopy(1)In different humidity environment,with the monitoring time increases,the transmission and redshift of the resonance peak of U87 cell-THz metamaterial increases and decreases,respectively.But when the environmental humidity increases from 3% to 7%,the duration of the resonance peak increases in each phase.(2)In the same humidity environment,the THz metamaterials with different resonance frequency continuously monitored U87 cells,and the duration of resonance peak is the same or very similar in each phase.(3)The average maximum height of single adherent U87 cell was 5.033 ?M.With the decrease of the thickness of PBS liquid layer,the transmission and redshift of the low-frequency resonance peak of THz metamaterial increases and decreases,respectively.Conclusion:1.Correlation between prognosis of multiple malignant tumors and tumor microenvironmentStromal score or immune score based on tumor microenvironment can evaluate the prognosis of 6 tumor types,which provides a new index for prognosis evaluation of tumor patients.BP,CC,MF,and the main central nodes in PPI network,involved in the common differentially expressed genes of stromal and immune score groups,have high consistency,indicating that at the gene level,different score groups have high similarity in the evaluation of tumor prognosis.The genes related to the prognosis of related tumor and expressed significantly different in normal and tumor tissues were identified,including integrin family,interleukin family and IGF system,which laid a foundation for screening potential tumor prognosis markers based on tumor microenvironment.2.Construction and methodology evaluation of SERS aptsensor based on enhanced substrate and silver nanoprobeQ-SERS substrate with good reproducibility and enhancement was successfully selected from five commercial SERS active substrates as the capture substrate of SERS aptsensor.AgNPs were successfully prepared and screened,which can be used to construct SERS nanotags and generate the strongest SERS signal in combination with Q-SERS substrates coated with aptamers.Finally,the SERS aptsensor was successfully constructed by combining the aptamer coated enhancement substrate and silver nanoprobe to form a sandwich structure.The SERS aptsensor has good sensitivity,specificity and stability,and can be used for the detection of clinical samples.This method can not only be extended to other detection platforms,but also be used to detect other proteins by replacing with different aptamers.3.Preparation,simulation and evaluation of cell activity and water content of silicon-based THz matematerial sensorIn this study,the silicon-based THz matematerial was designed and prepared,that can be used for the detection of monolayer cells.Compared with the conventional cell suspension detection,it greatly improves the sensitivity of cell detection.By using the change of the transmission and frequency of the resonance peak of THz metamaterials,the living state and water content of cells can be monitored in real time.At the same time,THz matematerial sensor can evaluate the effect of different humidity on cells.It provides a necessary reference for further detection of living cells,bacteria and liquid substances in the dry environment,especially in the control of detection time and humidity.