Evaluation Of Screening Efficacy Of Combined Test Indicators For Pancreatic Cancer And A Novel Method For CtDNA Detection Based On Terahertz Technology

BackgroundAmong various types of tumors,pancreatic cancer is highly malignant with a low five-year survival rate,and patients in early stage often delay getting medical attention due to insidous symptoms.The current diagnostic method mainly relies on pathological examination or imaging examination,which is expensive and invasive,and hardly used as a screening method.The most valuable tumor marker for pancreatic cancer is CA19-9,but its sensitivity and specificity are limited and cannot cater for the early diagnosis of pancreatic cancer.Combined testing of multiple tumor markers can improve the screening efficacy of tumors,but blinded combined testing increases the medical burden to patients.During the progression of pancreatic cancer,the enlarged tumor compresses the bile duct,which can cause abnormal liver function such as jaundice.In addition,inflammatory factors play an important role in the tumor microenvironment in promoting tumor development and metastasis.Therefore,making full use of the information of relevant indicators in routine blood and liver function test results,combined with CA19-9 is expected to improve the early screening efficacy of pancreatic cancer.In addition,the relationship between serum CA19-9,blood routine,liver function related indexes and pathological stage,clinical stage of pancreatic cancer patients was also investigated,which provided certain reference value for the early diagnosis of pancreatic cancer.Nucleic acid tumor markers such as Circulating tumor DNA（ctDNA）carry the same genetic information as solid tumor,which can reflect the characteristics of tumor at the molecular level and show abnormalities earlier than protein tumor markers,which can help in the early diagnosis of tumor.The current methods for ctDNA detection mainly include polymerase chain reaction（PCR）,digital PCR,and next-generation sequencing,which are costly and require professional personnel operation and sophisticated instruments,limiting its clinical application,new detection methods need to be explored.As a cutting-edge cross-disciplinary technology,Terahertz（THz）spectroscopy enables label-free characterization of nucleic acids,proteins,glucose and other substances with its unique properties.Highly sensitive detection of trace targets can be achieved using THz metamaterial technology,and graphene bas been used as THz wave modulation device due to its tunable Fermi level.Therefore,graphene metamaterials were fabricated,and the response mechanism of gold nanoparticles on graphene metamaterials was investigated,and a graphene-enhanced THz nanobiosensor was constructed for highly sensitive detection of ctDNA,which provided a new idea for the detection of nucleic acid-based tumor markers.Objectives1.To investigate the relationship between serum CA19-9,blood routine and liver function related indexes and pathological stage and clinical stage of pancreatic cancer patients and the value of their combined detection for the diagnosis of pancreatic cancer.2.To construct a graphene-enhanced THz nanobiosensor based on enzyme-free amplification for rapid and ultra sensitive detection of ctDNA.Methods1.185 patients with pancreatic cancer,91 patients with benign occupying lesions of the pancreas and 90 healthy individuals were recruited as study subjects,and their serum CA19-9,blood routine and liver function related indexes were statistically analyzed.2.The KRAS G12D mutant sequence was used as the target ctDNA,hybridization chain reaction（HCR）based on the surface of magnetic beads was constructed,probe and DNA hairpins compatible with the target ctDNA were designed,the probe was modified on the surface of magnetic beads,with the introduction of target ctDNA,one end of ctDNA could bind to the probe and the other end would initiate HCR,and the streptavidin-modified gold nanoparticles could bind to the biotin sites on the HCR product.The feasibility of the reaction was verified using non-denaturing polyacrylamide gel electrophoresis,zeta potential and transmission electron microscopy.3.Graphene metamaterial was designed and fabricated for the detection of the HCR products,quality of graphene was characterized using Raman spectroscopy,experimental and simulation studies on the THz signal response of gold nanoparticles on the surface of graphene metamaterials was carried out,and mechanism of gold nanoparticles enhancing the THz signal response of graphene metamaterials was elaborated.4.The above two modules were integrated to construct a HCR-based THz graphene metamaterial biosensor for the detection of target ctDNA.After optimizing the experimental conditions,the sensitivity and specificity of thebio sensor were characterized,and the target ctDNA recovery experiments in commercial human serum were carried out.ctDNA was extracted from clinical plasma and detected using the proposed biosensor,results were compared with those of qPCR to verify the consistency.Results1.The levels of PLR,NLR,CA19-9,ALP,and DBIL in the pancreatic cancer group were higher than those in the healthy group and the group with benign occupying lesions of the pancreas;levels of CA19-9,ALP,and DBIL were higher in patients with early stage（stage I）pancreatic cancer than those with benign occupying lesions;the combined detection of CA19-9,PLR,ALP,and DBIL could improve the screening efficacy of pancreatic cancer.The relative expression level of plasma ctDNA in pancreatic cancer patients was significantly higher than that in healthy people,suggested that ctDNA has the potential for early diagnosis of pancreatic cancer.2.Results of PAGE confirmed the feasibility of the constructed HCR strategy on the surface of magnetic beads.The zeta potential on the surface of the beads after the occurrence of HCR（-36.97±0.80 m V）was significantly lower than that on the surface of the blank beads（-21.67±0.96 m V）,which confirmed the occurrence of HCR on the surface of the beads.TEM showed that string bead-like gold nanoparticles were attached to the surface of the beads,which proved that the gold nanoparticles were connected to the beads through the HCR products.3.Graphene metamaterial chips were fabricated,and the transferred graphene was characterized by Raman spectroscopy,obvious G peak at 1586 cm^-1 and 2D peak at 2644 cm^-1were observed,and the intensity ratio of 2D peak to G peak was greater than 2,which proved fine quality of monolayer graphene.4.For the experimental study,as the mass of gold nanoparticles deposited on the surface of graphene metamaterial increased from 1 ng to 50 ng,the resonance peak of the metamaterial blue-shifted,and the resonance gradually enhanced,the transmittance at the resonance peak gradually decreased.For the simulation study,as the Fermi level of graphene moved from-0.07 e V to-0.01 e V towards the Dirac point,the resonance peak of the metamaterial blue-shifted and the resonance gradually enhanced,the transmittance at the resonance peak gradually decreased.5.The concentration of ctDNA molecules and the resonance amplitude variation rate of its products on THz graphene metamaterials had a good linear relationship in the range of 1fM-10 pM,with the linear equationΔT/T0（%）=3.096Log C+11.504,R²=0.9961,and the limit of detection was 0.22 fM.The resonance amplitude variation rate of the target ctDNA was significantly higher than that of KRAS wild-type sequences,KRAS G13D mutant sequences and KRAS G12C mutant sequences.Target ctDNA samples of 10 fM,100 fM,1pM and 10 pM in commercial human serum configuration were measured with recovery rate ranging from 97.36%to 104.63%and relative standard deviation（RSD）ranging from 5.17%to 11.24%.The proposed biosensor was used to detect target ctDNA extracted from clinical plasma,which was in good agreement with the results of qPCR（R²=0.9522）,indicating that the proposed biosensor can preliminarily distinguish pancreatic cancer patients from healthy people.Conclusions1.CA19-9,blood routine and liver function indexes were closely related to the pathological stage and clinical stage of pancreatic cancer,and the combined detection of CA19-9,PLR,ALP and DBIL can improve the screening efficacy of pancreatic cancer and have certain guiding significance for the early diagnosis and prognostic judgment of pancreatic cancer,but it is still far from clinical application,while ctDNA has the potential to realize the early diagnosis of pancreatic cancer.2.The mechanism of gold nanoparticles enhancing the response of graphene metamaterial was clarified,electrons transferred from gold nanoparticles to graphene as they interacted,which leaded to the shift of the Fermi level of graphene to the Dirac point,thus reducing the carrier concentration of graphene,and the reduction of graphene conductivity resulted in the enhancement of the resonance peak of the metamaterial,so that the trace detection of the target could be achieved by the change of THz resonance peak intensity.3.A graphene-enhanced THz nanobiosensor was constructed by combining the HCR amplification strategy on the surface of magnetic bead with the graphene metamaterial chip,realizing the highly sensitive and rapid detection of ctDNA.The biosensor had a good linear range and detection performance,and can effectively distinguish pancreatic cancer patients from healthy people,providing a new idea for the detection of nucleic acid tumor markers...